Use of (−) (3-trihalomethylphenoxy) (4-halophenyl) acetic acid derivatives for treatment of hyperuricemia

ABSTRACT

The present invention provides the use of (−)(3-trihalomethylphenoxy)(4-halophenyl)acetic acid derivatives and compositions in the treatment of insulin resistance, Type 2 diabetes, hyperlipidemia and hyperuricemia.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/382,186, filed Mar. 4, 2003, now U.S. Pat. No. 7,576,131, which is acontinuation-in-part of U.S. patent application Ser. No. 09/703,487,filed Oct. 31, 2000, now U.S. Pat. No. 6,646,004, which is acontinuation of U.S. patent application Ser. No. 09/325,997 filed onJun. 4, 1999, now U.S. Pat. No. 6,262,118; said U.S. patent applicationSer. No. 10/382,186, filed Mar. 4, 2003, now U.S. Pat. No. 7,576,131, isalso a continuation-in-part of U.S. patent application Ser. No.09/585,907, filed on Jun. 2, 2000, now U.S. Pat. No. 6,613,802, which isa continuation-in-part of U.S. patent application Ser. No. 09/325,997,filed on Jun. 4, 1999, now U.S. Pat. No. 6,262,118; said U.S. patentapplication Ser. No. 10/382,186, filed Mar. 4, 2003, now U.S. Pat. No.7,576,131, is also a continuation-in-part of U.S. patent applicationSer. No. 09/724,788, filed Nov. 28, 2000, now U.S. Pat. No. 6,624,194,which is a continuation in part of U.S. patent application Ser. No.09/585,907, filed on Jun. 2, 2000, now U.S. Pat. No. 6,613,802, which isa continuation-in-part of U.S. patent application Ser. No. 09/325,997,filed on Jun. 4, 1999, now U.S. Pat. No. 6,262,118. The contents ofthese priority applications are each herein individually incorporated byreference for all purposes.

FIELD OF THE INVENTION

The present invention relates to the use of(−)(3-trihalomethylphenoxy)(4-halophenyl)acetic acid derivatives andcompositions in the treatment of insulin resistance, Type 2 diabetes,hyperlipidemia and hyperuricemia.

BACKGROUND OF THE INVENTION

Diabetes mellitus, commonly called diabetes, refers to a disease processderived from multiple causative factors and characterized by elevatedlevels of plasma glucose, referred to as hyperglycemia. See, e.g.,LeRoith, D. et al., (eds.), DIABETES MELLITUS (Lippincott-RavenPublishers, Philadelphia, Pa. U.S.A. 1996), and all references citedtherein. According to the American Diabetes Association, diabetesmellitus is estimated to affect approximately 6% of the worldpopulation. Uncontrolled hyperglycemia is associated with increased andpremature mortality due to an increased risk for microvascular andmacrovascular diseases, including nephropathy, neuropathy, retinopathy,hypertension, cerebrovascular disease and coronary heart disease.Therefore, control of glucose homeostasis is a critically importantapproach for the treatment of diabetes.

There are two major forms of diabetes: Type 1 diabetes (formerlyreferred to as insulin-dependent diabetes or IDDM); and Type 2 diabetes(formerly referred to as non-insulin dependent diabetes or NIDDM).

Type 1 diabetes is the result of an absolute deficiency of insulin, thehormone which regulates glucose utilization. This insulin deficiency isusually characterized by β-cell destruction within the Islets ofLangerhans in the pancreas, which usually leads to absolute insulindeficiency. Type 1 diabetes has two forms: Immune-Mediated DiabetesMellitus, which results from a cellular mediated autoimmune destructionof the β cells of the pancreas; and Idiopathic Diabetes Mellitus, whichrefers to forms of the disease that have no known etiologies.

Type 2 diabetes is a disease characterized by insulin resistanceaccompanied by relative, rather than absolute, insulin deficiency. Type2 diabetes can range from predominant insulin resistance with relativeinsulin deficiency to predominant insulin deficiency with some insulinresistance. Insulin resistance is the diminished ability of insulin toexert its biological action across a broad range of concentrations. Ininsulin resistant individuals, the body secretes abnormally high amountsof insulin to compensate for this defect. When inadequate amounts ofinsulin are present to compensate for insulin resistance and adequatelycontrol glucose, a state of impaired glucose tolerance develops. In asignificant number of individuals, insulin secretion declines furtherand the plasma glucose level rises, resulting in the clinical state ofdiabetes. Type 2 diabetes can be due to a profound resistance to insulinstimulating regulatory effects on glucose and lipid metabolism in themain insulin-sensitive tissues: muscle, liver and adipose tissue. Thisresistance to insulin responsiveness results in insufficient insulinactivation of glucose uptake, oxidation and storage in muscle andinadequate insulin repression of lipolysis in adipose tissue and ofglucose production and secretion in liver. In Type 2 diabetes, freefatty acid levels are often elevated in obese and some non-obesepatients and lipid oxidation is increased.

Premature development of atherosclerosis and increased rate ofcardiovascular and peripheral vascular diseases are characteristicfeatures of patients with diabetes. Hyperlipidemia is an importantprecipitating factor for these diseases.

Hyperlipidemia is a condition generally characterized by an abnormalincrease in serum lipids in the bloodstream and is an important riskfactor in developing atherosclerosis and heart disease. For a review ofdisorders of lipid metabolism, see, e.g., Wilson, J. et al., (ed.),Disorders of Lipid Metabolism, Chapter 23, Textbook of Endocrinology,9^(th) Edition, (W.B. Sanders Company, Philadelphia, Pa. U.S.A. 1998;this reference and all references cited therein are herein incorporatedby reference). Serum lipoproteins are the carriers for lipids in thecirculation. They are classified according to their density:chylomicrons; very low-density lipoproteins (VLDL); intermediate densitylipoproteins (IDL); low density lipoproteins (LDL); and high densitylipoproteins (HDL). Hyperlipidemia is usually classified as primary orsecondary hyperlipidemia. Primary hyperlipidemia is generally caused bygenetic defects, while secondary hyperlipidemia is generally caused byother factors, such as various disease states, drugs, and dietaryfactors. Alternatively, hyperlipidemia can result from both acombination of primary and secondary causes of hyperlipidemia. Elevatedcholesterol levels are associated with a number of disease states,including coronary artery disease, angina pectoris, carotid arterydisease, strokes, cerebral arteriosclerosis, and xanthoma.

Dyslipidemia, or abnormal levels of lipoproteins in blood plasma, is afrequent occurrence among diabetics, and has been shown to be one of themain contributors to the increased incidence of coronary events anddeaths among diabetic subjects (see, e.g., Joslin, E. Ann. Chim. Med.(1927) δ: 1061-1079). Epidemiological studies since then have confirmedthe association and have shown a several-fold increase in coronarydeaths among diabetic subjects when compared with nondiabetic subjects(see, e.g., Garcia, M. J. et al., Diabetes (1974) 23: 105-11 (1974); andLaakso, M. and Lehto, S., Diabetes Reviews (1997) 5(4): 294-315).Several lipoprotein abnormalities have been described among diabeticsubjects (Howard B., et al., Arthrosclerosis (1978) 30: 153-162).

Previous studies from the 1970's have demonstrated the effectiveness ofracemic2-acetamidoethyl(4-chlorophenyl)(3-trifluoromethylphenoxy)acetate (alsoknown as “halofenate”) as a potential therapeutic agent to treat Type 2diabetes, hyperlipidemia and hyperuricemia (see, e.g., Bolhofer, W.,U.S. Pat. No. 3,517,050; Jain, A. et al., N. Eng. J. Med. (1975) 293:1283-1286; Kudzma, D. et al., Diabetes (1977) 25: 291-95; Kohl, E. etal., Diabetes Care (1984) 7: 19-24; McMahon, F. G. et al., Univ. Mich.Med. Center J. (1970) 36: 247-248; Simori, C. et al., Lipids (1972) 7:96-99; Morgan, J. P. et al., Clin. Pharmacol. Therap. (1971) 12:517-524, Aronow, W. S. et al., Clin. Pharmacol Ther (1973) 14: 358-365and Fanelli, G. M. et al., J. Pharm. Experimental Therapeutics (1972)180:377-396). In these previous studies, the effect of racemichalofenate on diabetes was observed when combined with sulfonylureas. Aminimal effect on glucose was observed in patients with diabetes treatedwith racemic halofenate alone. However, significant side effects werenoted including gastrointestinal bleeding from stomach and peptic ulcers(see, e.g., Friedberg, S. J. et al., Clin. Res. (1986) Vol. 34, No. 2:682A).

In addition, there were some indications of drug-drug interactions ofracemic halofenate with agents such as warfarin sulfate (also referredto as 3-(alpha-acetonylbenzyl)-4-hydroxycoumarin or Coumadin™ (DupontPharmaceuticals, E.I. Dupont de Nemours and Co., Inc., Wilmington, Del.U.S.A.) (see, e.g., Vesell, E. S. and Passantanti, G. T., Fed. Proc.(1972) 31(2): 538). Coumadin™ is an anticoagulant that acts byinhibiting the synthesis of vitamin K dependent clotting factors (whichinclude Factors II, VII, IX, and X, and the anticoagulant proteins C andS). Coumadin™ is believed to be stereospecifically metabolized byhepatic microsomal enzymes (the cytochrome P450 enzymes). The cytochromeP450 isozymes involved in the metabolism of Coumadin include 2C9, 2C19,2C8, 2C18, 1A2, and 3A4. 2C9 is likely to be the principal form of humanliver P450 which modulates in vivo drug metabolism of several drugsincluding the anticoagulant activity of Coumadin™ (see, e.g., Miners, J.O. et al., Bri. J. Clin. Pharmacol. (1998) 45: 525-538).

Drugs that inhibit the metabolism of Coumadin™ result in a furtherdecrease in vitamin K dependent clotting factors that preventscoagulation more than desired in patients receiving such therapy (i.e.,patients at risk for pulmonary or cerebral embolism from blood clots intheir lower extremities, heart or other sites). Simple reduction of thedose of anticoagulant is often difficult as one needs to maintainadequate anticoagulation to prevent blood clots from forming. Theincreased anticoagulation from drug-drug interaction results in asignificant risk to such patients with the possibility of severebleeding from soft tissue injuries, gastrointestinal sites (i.e.,gastric or duodenal ulcers) or other lesions (i.e., aortic aneurysm).Bleeding in the face of too much anticoagulation constitutes a medicalemergency and can result in death if it is not treated immediately withappropriate therapy.

Cytochrome P450 2C9 is also known to be involved in the metabolism ofseveral other commonly used drugs, including dilantin, sulfonylureas,such as tolbutamide and several nonsteroidal anti-inflammatory agents,such as ibuprofen. Inhibition of this enzyme has the potential to causeother adverse effects related to drug-drug interactions, in addition tothose described above for Coumadin™ (see, e.g., Pelkonen, O. et al.,Xenobiotica (1998) 28: 1203-1253; Linn, J. H. and Lu, A. Y., Clin.Pharmacokinet. (1998) 35(5): 361-390).

Treatment with racemic halofenate has been associated with an increasedrisk of gastrointestinal side effects such as upper gastrointestinalulcers and bleeding. (see, e.g., Friedberg, S. J. et al., Clin. Res.(1986) Vol. 34, No. 2: 682A). Non-steroidal antiinflammatory drugs(NSAIDs, e.g., ibuprofen, indomethacin, naproxen, aspirin) which arenon-selective inhibitors of the cyclooxygenases I and II (COX I andCOX-II, respectively) are often associated with such side effects. Theseagents control the biosynthesis of prostaglandins by inhibitingcyclooxygenases which are synthases responsible for the formation ofprostaglandins from arachidonic acid. Inhibition of prostaglandinproduction is anti-inflammatory. However, prostaglandins produced by theaction of COX-I are primarily involved in inhibiting gastric secretionand increasing mucosal blood flow. Such COX-I inhibitors have a highpotential for raising adverse side effects such as attacks on gastricmucosa and kidney and limited clinical utility. Moreover, the druginteractions affecting coumadin metabolism in the circumstance of anadverse drug effect on gastrointestinal bleeding is of compoundedconcern. In distinction to inhibition of the COX-I enzyme, theinhibition of the COX-II enzyme is associated with the more beneficialanti-inflammatory effects of the NSAIDs. Agents which target the COX-IIenzyme are therefore preferred antiinflammatory agents for mostpurposes.

Solutions to the above difficulties and deficiencies are needed beforehalofenate becomes effective for routine treatment of insulinresistance, Type 2 diabetes, hyperlipidemia and hyperuricemia. Thepresent invention fulfills this and other needs by providingcompositions and methods for alleviating insulin resistance, Type 2diabetes, hyperlipidemia and hyperuricemia, while presenting a betteradverse effect profile, particularly with respect to gastrointestinalbleeding from stomach and peptic ulcers and adverse drug interactions.

SUMMARY OF THE INVENTION

This present invention provides a method of modulating blood ormetabolic disorders in a mammal such as Type 2 diabetes, dyslipidemia,and hyperuricemia in a mammal by administering compounds with a reducedpotential to cause gastrointestinal or drug interaction side effects.The method comprises administering to the mammal a therapeuticallyeffective amount of the (−) stereoisomer of a compound of Formula I,

wherein R is a member selected from the group consisting of a hydroxy,lower aralkoxy, di-lower alkylamino-lower alkoxy, lower alkanamido loweralkoxy, benzamido-lower alkoxy, ureido-lower alkoxy, N′-loweralkyl-ureido-lower alkoxy, carbamoyl-lower alkoxy, halophenoxysubstituted lower alkoxy, carbamoyl substituted phenoxy, carbonyl-loweralkylamino, N,N-di-lower alkylamino-lower alkylamino, halo substitutedlower alkylamino, hydroxy substituted lower alkylamino, loweralkanolyloxy substituted lower alkylamino, ureido, and loweralkoxycarbonylamino; and X is a halogen; or a pharmaceuticallyacceptable salt thereof, wherein the administered compound issubstantially free of its (+) stereoisomer.

Some such methods further comprise administering a (−) stereoisomer of acompound of Formula II:

wherein R² is a member selected from the group consisting ofphenyl-lower alkyl, lower alkanamido-lower alkyl, and benzamido-loweralkyl.

Some such methods comprise administering a (−) stereoisomer of acompound of Formula III:

The preferred compound of Formula III is known as “(−)2-acetamidoethyl4-chlorophenyl-(3-trifluoromethylphenoxy)-acetate” or “(−) halofenate.”

The present invention further provides a method for modulating insulinresistance in a mammal. This method comprises administering to themammal a therapeutically effective amount of the (−) stereoisomer of acompound of Formula I wherein the compound is substantially free of its(+) stereoisomer. Some such methods comprise a (−) stereoisomer of acompound of Formula II. Some such methods comprise a (−) stereoisomercompound of Formula III.

The present invention further provides a method of alleviatinghyperlipidemia in a mammal. This method comprises administering to themammal a therapeutically effective amount of a (−) stereoisomer of acompound of Formula I wherein the compound is substantially free of its(+) stereoisomer. In some such methods, the compound is a (−)stereoisomer of a compound of Formula II. In other such methods, thecompound is a (−) stereoisomer of a compound of Formula III.

The present invention further provides a method of modulatinghyperuricemia in a mammal. This method comprises administering to themammal a therapeutically effective amount of a compound of Formula Iwherein the compound is substantially free of its (+) stereoisomer. Insome such methods, the compound is a (−) stereoisomer of a compound ofFormula II. In other such methods, the compound is a (−) stereoisomer ofa compound of Formula III.

The present invention also provides the (−) stereoisomers of compoundsof Formula I, II, or III. The present invention also providescompositions of the (−) stereoisomers of compounds of Formula I, II, orIII which are substantially free of the corresponding (+) stereoisomersof the compound. In another embodiment, the invention provides (−)stereoisomers of the compounds of Formula I, II, or III which have anIC₅₀ for the COX-1 enzyme which is a least two-fold greater than theIC₅₀ of its (+) stereoisomer. In other embodiments, the IC50 is at least3-fold greater or at least 4-fold greater than the IC₅₀ of thecorresponding (+) stereoisomer. In other embodiments, the inventionprovides a (−) stereoisomer of a compound of Formula I, II, or IIIwherein the IC50 exceeds the solubility limit of the compound. In otheraspects, the invention provides methods of treating diabetes,dyslipidemia, hyperlipidemia, or hyperuricemia by administering suchinventive compounds.

In another embodiment, the invention provides (−) stereoisomers of thecompounds of Formula I, II, or III which have an IC₅₀ for cytochromeP450 2C9 that is a least five-fold greater, 10-fold greater, or 20-foldgreater than the IC₅₀ of the corresponding (+) stereoisomer. In otheraspects, the invention provides methods of treating diabetes,hyperlipidemia, and hyperuricemia by administering such inventivecompounds.

In another embodiment, the invention provides (−) stereoisomers of thecompounds of Formula I, II, or III which have an IC₅₀ for cytochromeP450 2C9 that is a least five-fold greater than the IC₅₀ of thecorresponding (+) stereoisomer and which also have an IC₅₀ forinhibition of the COX-1 enzyme which is at least 3-fold greater than theIC₅₀ of the corresponding (+) stereoisomer. In other aspects, theinvention provides methods of treating diabetes, dyslipidemia,hyperlipidemia, or hyperuricemia by administering such inventivecompounds.

The present invention also provides pharmaceutical compositions. Thepharmaceutical compositions comprise a pharmaceutically acceptablecarrier and a therapeutically effective amount of a (−) stereoisomer ofa compound of Formula I, Formula II or Formula III wherein thecomposition is substantially free of the (+) isomer of the compound ofFormula I, II, or III.

In another preferred embodiment, the compositions are in unit doseformat and comprise a pharmaceutically acceptable carrier and atherapeutically effective amount of a compound of Formula I, Formula IIor Formula III in which the compound is a (−) stereoisomer and theamount of the (−) isomer is insufficient to cause adverse effects on thegastrointestinal tract such as gastrointestinal bleeding or mucosalerosion associated with the inhibition of the COX-1 enzyme and whereinthe composition is substantially free of the (+) isomer of the compoundof Formula I, II, or III.

In other embodiments, the compositions are in unit dose format andcomprise a pharmaceutically acceptable carrier and a therapeuticallyeffective amount of a compound of Formula I, Formula II or Formula IIIin which the compound is a (−) stereoisomer that has at least two foldgreater IC₅₀ for inhibiting the COX-1 enzyme than the corresponding (+)isomer. In a further embodiment, the COX-1 IC₅₀ for the (−) stereoisomeris greater than 2 mM. In one embodiment, the (−) stereoisomer at atherapeutic dose level is associated with a substantially lowerincidence of compound-related gastrointestinal ulcers than that of acomparable therapeutic level of the (+) stereoisomer. In preferred suchembodiments, the composition is substantially free of the (+) isomer ofthe compound of Formula I, II, or III.

In another embodiment, the compositions are in unit dose format andcomprise a pharmaceutically acceptable carrier and a therapeuticallyeffective amount of a compound of Formula I, Formula II or Formula IIIin which the compound is a (−) stereoisomer and the amount of the (−)isomer is insufficient to cause adverse effects associated with theinhibition of cytochrome P450 C29. In preferred such embodiments, thecomposition is substantially free of the (+) isomer of the compound ofFormula I, II, or III.

In other embodiments, the compositions are in unit dose format andcomprise a pharmaceutically acceptable carrier and a therapeuticallyeffective amount of a compound of Formula I, Formula II or Formula IIIin which the compound is a (−) stereoisomer that has at least afive-fold greater IC₅₀ for inhibiting cytochrome P450 2C9 than thecorresponding (+) isomer wherein the composition is substantially freeof the (+) isomer of the compound of Formula I, II, or III. In furtherembodiments, the cytochrome P450 2C9 IC₅₀ for the (−) stereoisomer isgreater than 1, 5, or 10 micromolars. In another embodiment, the (−)stereoisomer is a compound which does not cause adverse drug-druginteractions with coumadin at therapeutic concentrations of the (−)stereoisomer.

In preferred embodiments of the above, the mammal is a human; the COX-1enzyme is the human enzyme; and the cytochrome P450 2C9 enzyme is thehuman enzyme.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the inhibition of cytochrome P450 2C9 (CYP2C9) activity byracemic halofenic acid, (−) halofenic acid and (+) halofenic acid. Thehydroxylation of tolbutamide was measured in the presence of increasingconcentrations of these compounds. Racemic halofenic acid inhibited CYP2C9 activity with an IC50 of 0.45 μM and (+) halofenic acid inhibitedCYP 2C9 with an IC50 of 0.22 μM. In contrast, the (−) halofenic acid was20-fold less potent with an apparent IC50 of 3.5 μM.

FIG. 2 shows the time course of glucose-lowering following a single oraldose of racemic halofenate, (−) enantiomer of halofenate or (+)enantiomer of halofenate at 250 mg/kg in diabetic ob/ob mice. The (−)enantiomer showed the most rapid onset of action and the longestduration of action. The decrease in glucose was significant (p<0.05) forthe (−) enantiomer compared to control for all points from 3 to 24hours. Racemic halofenate and the (+) enantiomer were also significant(p<0.05) for all points from 4.5 to 24 hours. The plasma glucose at 24hours was 217±16.4 mg/dl in animals treated with the (−) enantiomer,compared to 306±28.5 mg/dl and 259.3±20.8 mg/dl for animals treated withthe (+) enantiomer and the racemate, respectively. The plasma glucose inthe vehicle treated controls was 408±16.2 mg/dl at 24 hours. The (−)enantiomer was more effective and significantly different (p<0.05) fromthe (+) enantiomer at both the 3 hour and 24 hour time points.

FIG. 3 shows the ability of racemic halofenate and both the (−) and (+)enantiomers of halofenate to lower plasma glucose in diabetic ob/ob micefollowing daily oral administration. The racemate was given at a dose of250 mg/kg/day and the enantiomers were given at doses of 125 mg/kg/dayand 250 mg/kg/day. Significant decreases in glucose levels relative tocontrol animals were observed in animals treated with racemic halofenateand both the (−) and (+) enantiomers. At the low dose (125 mg/kg) oftreatment with the (−) and (+) enantiomers, the (−) enantiomer wassignificant at 6, 27 and 30 hours whereas the (+) enantiomer wassignificant at only 6 and 27 hours.

FIG. 4 shows the plasma insulin levels in the ob/ob mice treated withracemic halofenate and both the (−) and (+) enantiomers of halofenate indiabetic ob/ob mice following daily oral administration. The racematewas given at a dose of 250 mg/kg/day and the enantiomers were given atdoses of 125 mg/kg/day and 250 mg/kg/day. Relative to the vehiclecontrol, insulins were lower in the animals treated with either theracemate or either of the enantiomers of halofenate. At the high dose,the greatest extent of reduced plasma insulin was noted at 27 and 30hours in animals treated with both the (−) and (+) enantiomers ofhalofenate following two days of treatment.

FIG. 5 shows plasma glucose levels following an overnight fast in ob/obmice after 5 days treatment with vehicle, racemic halofenate at 250mg/kg/day, (−) enantiomer of halofenate at 125 mg/kg/day and 250mg/kg/day or (+) enantiomer of halofenate at 125 mg/kg/day or 250mg/kg/day. The control animals were hyperglycemic with plasma glucoselevels of 185.4±12.3 mg/dl. All of the animals treated with halofenateshowed significant (p<0.01) reductions in glucose. The high doses ofboth enantiomers lowered the glucose to near normal levels at 127.3±8.0mg/dl and 127.2±9.7 mg/dl for the (−) enantiomer and (+) enantiomertreated animals, respectively.

FIG. 6 shows the overnight fasting plasma insulin levels in the ob/obmice treated with vehicle, racemic halofenate at 250 mg/kg/day, (−)enantiomer at 125 mg/kg/day and 250 mg/kg/day or (+) enantiomer ofhalofenate at 125 mg/kg/day or 250 mg/kg/day for 5 days. Significantlylower plasma insulins were observed in animals receiving both doses of(−) enantiomer. The low dose of (+) enantiomer of halofenate did notlower plasma insulin, although the high dose of the (+) enantiomerresulted in a decrease in plasma insulin.

FIG. 7A shows plasma glucose levels following an oral glucose challengein Zucker fatty rats, a model of insulin resistance and Impaired GlucoseTolerance. These animals were treated with either a vehicle control,racemic halofenate, (−) halofenate or (+) halofenate 5.5 hours prior tothe glucose challenge. The racemate was given at 100 mg/kg and both ofthe enantiomers were given at 50 and 100 mg/kg. In the control animalsthe glucose rose to >250 mg/dl 30 minutes after the challenge, a clearindication of impaired glucose tolerance. The plasma glucose was reducedin rats that had received racemic halofenate, especially between 30-60minutes after the challenge. Animals that received the (−) halofenate at100 mg/kg had the greatest degree of glucose-lowering of all the treatedanimals. Animals treated with the (−) halofenate had lower glucoselevels that persisted at 90-120 minutes, compared to those rats treatedwith the racemate or (+) halofenate. FIG. 7B compares the incrementalarea under the curve (AUC) for the animals in each group. Significantchanges (p<0.05) were noted in the groups treated with both doses of the(−) halofenate. Although the AUC was lower in the other groups relativeto the control, the changes were not significant.

FIG. 8 shows the results of a short insulin tolerance test in Zuckerfatty rats that were treated with either a vehicle control, (−)halofenate (50 mg/kg/day) or (+) halofenate (50 mg/kg/day) for 5 days.This test is a measure of the insulin sensitivity of the test animals,the slope of the decline in glucose representing a direct measure ofinsulin responsiveness. The (−) halofenate-treated animals weresignificantly more insulin sensitive than the vehicle-treated (p<0.01)or the (+) halofenate-treated (p<0.05) animals.

FIG. 9A shows plasma cholesterol levels in Zucker Diabetic Fatty ratstreated for 13 days with racemic halofenate, (−) enantiomer or (+)enantiomer at 50 mg/kg/day, 25 mg/kg/day or 25 mg/kg/day, respectively,relative to a vehicle treated control group. In both the (−) enantiomerand racemate treated animals, the plasma cholesterol declined withtreatment. The cholesterol in the (+) enantiomer treated animalsremained relatively constant, whereas cholesterol rose in the controlanimals. FIG. 9B compares the differences in plasma cholesterol betweenthe control group and the treated groups. The (−) enantiomer was themost active of the species tested.

FIG. 10A shows plasma cholesterol levels in Zucker Diabetic Fatty ratstreated for 14 days with either (−) enantiomer or (+) enantiomer ofhalofenate at either 12.5 mg/kg/day (Low dose) or 37.5 mg/kg/day (Highdose) relative to a vehicle treated control group. In the animalstreated with the high dose, the (−) enantiomer resulted in the greatestextent of cholesterol lowering. FIG. 10B compares the differences inplasma cholesterol between the control and treated groups. There weresignificant differences in the animals treated with the (−) enantiomerafter 7 days at the low dose and after both 7 and 14 days at the highdose. The (+) enantiomer showed significance only after 7 days oftreatment at the high dose.

FIG. 11A shows plasma triglyceride levels in Zucker Diabetic Fatty ratstreated with either (−) enantiomer or (+) enantiomer at either 12.5mg/kg/day (Low dose) or 37.5 mg/kg/day (High dose) relative to a vehicletreated control group. Animals treated with the high dose of the (−)enantiomer had the lowest triglyceride levels of all the treatmentgroups. FIG. 11B compares the differences in plasma triglyceride betweenthe control and treated groups. At 7 days, the high dose of both the (+)and (−) enantiomers showed significant lowering of plasma triglyceride.

FIG. 12 shows plasma glucose levels in Zucker Diabetic Fatty ratstreated with vehicle, (−) halofenate or (+) halofenate at day 0, day 2and day 3. Treatment with (−) halofenate significantly reduced plasmaglucose concentrations as compared to vehicle-treated animals.

FIG. 13 shows plasma glucose concentrations in a control group ofC57BL/6J db/db mice versus in a group treated with (−) halofenate.Plasma glucose levels in the control group increased progressively asanimals aged, while the increase of plasma glucose levels in the (−)halofenate treated group was prevented or significantly delayed.

FIG. 14 shows plasma insulin levels in a control group of C57BL/6J db/dbmice versus in a group treated with (−) halofenate. Treatment with (−)halofenate maintained the plasma insulin concentration, while plasmainsulin in the control group decreased progressively.

FIG. 15 shows the percentage of non-diabetic mice in a control group ofC57BL/6J db/db mice versus in a group treated with (−) halofenate. About30% of mice in the (−) halofenate treated group did not develop diabetes(plasma glucose levels<250 mg/dl), while the entire control group did bythe age of 10 weeks.

FIG. 16 shows plasma triglyceride levels in a control group of C57BL/6Jdb/db mice versus in a group treated with (−) halofenate. Treatment with(−) halofenate alleviated hyperlipidemia, while there was no alleviationin the control group.

FIG. 17 shows the effect of (−) halofenate and (+) halofenate on plasmauric acid levels in oxonic acid induced hyperuricemic rats. Oraladministration of (−) halofenate significantly reduced plasma uric acidlevels. (+) halofenate also lowered plasma uric acid levels, but it wasnot statistically significant.

FIG. 18 shows the effect of ketoprofen, the positive control, racemichalofenate and MBX-102 ((−) halofenate) on inhibition of COX-1 in ahuman whole blood assay. (−) halofenate was much less effective than theracemate in inhibiting COX-1.

DEFINITIONS

The term “mammal” includes, without limitation, humans, domestic animals(e.g., dogs or cats), farm animals (cows, horses, or pigs), monkeys,rabbits, mice, and laboratory animals.

The term “insulin resistance” can be defined generally as a disorder ofglucose metabolism. More specifically, insulin resistance can be definedas the diminished ability of insulin to exert its biological actionacross a broad range of concentrations producing less than the expectedbiologic effect. (see, e.g., Reaven, G. M., J. Basic & Clin. Phys. &Pharm. (1998) 9: 387-406 and Flier, J. Ann Rev. Med. (1983) 34: 145-60).Insulin resistant persons have a diminished ability to properlymetabolize glucose and respond poorly, if at all, to insulin therapy.Manifestations of insulin resistance include insufficient insulinactivation of glucose uptake, oxidation and storage in muscle andinadequate insulin repression of lipolysis in adipose tissue and ofglucose production and secretion in liver. Insulin resistance can causeor contribute to polycystic ovarian syndrome, Impaired Glucose Tolerance(IGT), gestational diabetes, hypertension, obesity, atherosclerosis anda variety of other disorders. Eventually, the insulin resistantindividuals can progress to a point where a diabetic state is reached.The association of insulin resistance with glucose intolerance, anincrease in plasma triglyceride and a decrease in high-densitylipoprotein cholesterol concentrations, high blood pressure,hyperuricemia, smaller denser low-density lipoprotein particles, andhigher circulating levels of plaminogen activator inhibitor-1), has beenreferred to as “Syndrome X” (see, e.g., Reaven, G. M., Physiol. Rev.(1995) 75: 473-486).

The term “diabetes mellitus” or “diabetes” means a disease or conditionthat is generally characterized by metabolic defects in production andutilization of glucose which result in the failure to maintainappropriate blood sugar levels in the body. The result of these defectsis elevated blood glucose, referred to as “hyperglycemia.” Two majorforms of diabetes are Type 1 diabetes and Type 2 diabetes. As describedabove, Type 1 diabetes is generally the result of an absolute deficiencyof insulin, the hormone which regulates glucose utilization. Type 2diabetes often occurs in the face of normal or even elevated levels ofinsulin and can result from the inability of tissues to respondappropriately to insulin. Most Type 2 diabetic patients are insulinresistant and have a relative deficiency of insulin, in that insulinsecretion can not compensate for the resistance of peripheral tissues torespond to insulin. In addition, many Type 2 diabetics are obese. Othertypes of disorders of glucose homeostasis include Impaired GlucoseTolerance, which is a metabolic stage intermediate between normalglucose homeostasis and diabetes, and Gestational Diabetes Mellitus,which is glucose intolerance in pregnancy in women with no previoushistory of Type 1 or Type 2 diabetes.

The term “secondary diabetes” is diabetes resulting from otheridentifiable etiologies which include: genetic defects of β cellfunction (e.g., maturity onset-type diabetes of youth, referred to as“MODY,” which is an early-onset form of Type 2 diabetes with autosomalinheritance; see, e.g., Fajans S. et al., Diabet. Med. (1996) (9 Suppl6): S90-5 and Bell, G. et al., Annu. Rev. Physiol. (1996) 58: 171-86;genetic defects in insulin action; diseases of the exocrine pancreas(e.g., hemochromatosis, pancreatitis, and cystic fibrosis); certainendocrine diseases in which excess hormones interfere with insulinaction (e.g., growth hormone in acromegaly and cortisol in Cushing'ssyndrome); certain drugs that suppress insulin secretion (e.g.,phenyloin) or inhibit insulin action (e.g., estrogens andglucocorticoids); and diabetes caused by infection (e.g., rubella,Coxsackie, and CMV); as well as other genetic syndromes.

The guidelines for diagnosis for Type 2 diabetes, impaired glucosetolerance, and gestational diabetes have been outlined by the AmericanDiabetes Association (see, e.g., The Expert Committee on the Diagnosisand Classification of Diabetes Mellitus, Diabetes Care, (1999) Vol 2(Suppl 1): S5-19).

The term “halofenic acid” refers to the acid form of4-Chlorophenyl-(3-trifluoromethylphenoxy)-acetic acid.

The term “hyperinsulinemia” refers to the presence of an abnormallyelevated level of insulin in the blood.

The term “hyperuricemia” refers to the presence of an abnormallyelevated level of uric acid in the blood.

The term “secretagogue” means a substance or compound that stimulatessecretion. For example, an insulin secretagogue is a substance orcompound that stimulates secretion of insulin.

The term “hemoglobin” or “Hb” refers to a respiratory pigment present inerythrocytes, which is largely responsible for oxygen transport. Ahemoglobin molecule comprises four polypeptide subunits (two a chainsystems and two β chain systems, respectively). Each subunit is formedby association of one globin protein and one heme molecule which is aniron-protoporphyrin complex. The major class of hemoglobin found innormal adult hemolysate is adult hemoglobin (referred to as “HbA”; alsoreferred to HbA₀ for distinguishing it from glycated hemoglobin, whichis referred to as “HbA₁,” described infra) having α₂β₂ subunits. Tracecomponents such as HbA₂ (α₂δ₂) can also be found in normal adulthemolysate.

Among classes of adult hemoglobin HbAs, there is a glycated hemoglobin(referred to as “HbA₁,” or “glycosylated hemoglobin”), which may befurther fractionated into HbA_(1a1), HbA_(1a2), HbA_(1b), and HbA_(1c)with an ion exchange resin fractionation. All of these subclasses havethe same primary structure, which is stabilized by formation of analdimine (Schiff base) by the amino group of N-terminal valine in the βsubunit chain of normal hemoglobin HbA and glucose (or,glucose-6-phosphate or fructose) followed by formation of ketoamine byAmadori rearrangement.

The term “glycosylated hemoglobin” (also referred to as “HbA_(1c),”,“GHb”, “hemoglobin-glycosylated”, “diabetic control index” and“glycohemoglobin”; hereinafter referred to as “hemoglobin A_(1c)”)refers to a stable product of the nonenzymatic glycosylation of theβ-chain of hemoglobin by plasma glucose. Hemoglobin A_(1c) comprises themain portion of glycated hemoglobins in the blood. The ratio ofglycosylated hemoglobin is proportional to blood glucose level.Therefore, hemoglobin A_(1c) rate of formation directly increases withincreasing plasma glucose levels. Since glycosylation occurs at aconstant rate during the 120-day lifespan of an erythrocyte, measurementof glycosylated hemoglobin levels reflect the average blood glucoselevel for an individual during the preceding two to three months.Therefore determination of the amount of glycosylated hemoglobinHbA_(1c) can be a good index for carbohydrate metabolism control.Accordingly, blood glucose levels of the last two months can beestimated on the basis of the ratio of HbA_(1c) to total hemoglobin Hb.The analysis of the hemoglobin A_(1c) in blood is used as a measurementenabling long-term control of blood glucose level (see, e.g., Jain, S.,et al., Diabetes (1989) 38: 1539-1543; Peters A., et al., JAMA (1996)276: 1246-1252).

The term “symptom” of diabetes, includes, but is not limited to,polyuria, polydipsia, and polyphagia, as used herein, incorporatingtheir common usage. For example, “polyuria” means the passage of a largevolume of urine during a given period; “polydipsia” means chronic,excessive thirst; and “polyphagia” means excessive eating. Othersymptoms of diabetes include, e.g., increased susceptibility to certaininfections (especially fungal and staphylococcal infections), nausea,and ketoacidosis (enhanced production of ketone bodies in the blood).

The term “complication” of diabetes includes, but is not limited to,microvascular complications and macrovascular complications.Microvascular complications are those complications which generallyresult in small blood vessel damage. These complications include, e.g.,retinopathy (the impairment or loss of vision due to blood vessel damagein the eyes); neuropathy (nerve damage and foot problems due to bloodvessel damage to the nervous system); and nephropathy (kidney diseasedue to blood vessel damage in the kidneys). Macrovascular complicationsare those complications which generally result from large blood vesseldamage. These complications include, e.g., cardiovascular disease andperipheral vascular disease. Cardiovascular disease refers to diseasesof blood vessels of the heart. See. e.g., Kaplan, R. M., et al.,“Cardiovascular diseases” in HEALTH AND HUMAN BEHAVIOR, pp. 206-242(McGraw-Hill, New York 1993). Cardiovascular disease is generally one ofseveral forms, including, e.g., hypertension (also referred to as highblood pressure), coronary heart disease, stroke, and rheumatic heartdisease. Peripheral vascular disease refers to diseases of any of theblood vessels outside of the heart. It is often a narrowing of the bloodvessels that carry blood to leg and arm muscles.

The term “atherosclerosis” encompasses vascular diseases and conditionsthat are recognized and understood by physicians practicing in therelevant fields of medicine. Atherosclerotic cardiovascular disease,coronary heart disease (also known as coronary artery disease orischemic heart disease), cerebrovascular disease and peripheral vesseldisease are all clinical manifestations of atherosclerosis and aretherefore encompassed by the terms “atherosclerosis” and“atherosclerotic disease”.

The term “antihyperlipidemic” refers to the lowering of excessive lipidconcentrations in blood to desired levels.

The term “antiuricemic” refers to the lowering of excessive uric acidconcentrations in blood to desired levels.

The term “hyperlipidemia” refers to the presence of an abnormallyelevated level of lipids in the blood. Hyperlipidemia can appear in atleast three forms: (1) hypercholesterolemia, i.e., an elevatedcholesterol level; (2) hypertriglyceridemia, i.e., an elevatedtriglyceride level; and (3) combined hyperlipidemia, i.e., a combinationof hypercholesterolemia and hypertriglyceridemia.

The term “modulate” refers to the treating, prevention, suppression,enhancement or induction of a function or condition. For example, thecompounds of the present invention can modulate hyperlipidemia bylowering cholesterol in a human, thereby suppressing hyperlipidemia.

The term “treating” means the management and care of a human subject forthe purpose of combating the disease, condition, or disorder andincludes the administration of a compound of the present invention toprevent the onset of the symptoms or complications, alleviating thesymptoms or complications, or eliminating the disease, condition, ordisorder.

The term “preventing” means the management and care of a human subjectsuch that the onset of symptoms of a disease, condition or disorder doesnot occur.

The term “cholesterol” refers to a steroid alcohol that is an essentialcomponent of cell membranes and myelin sheaths and, as used herein,incorporates its common usage. Cholesterol also serves as a precursorfor steroid hormones and bile acids.

The term “triglyceride(s)” (“TGs”), as used herein, incorporates itscommon usage. TGs consist of three fatty acid molecules esterified to aglycerol molecule and serve to store fatty acids which are used bymuscle cells for energy production or are taken up and stored in adiposetissue.

Because cholesterol and TGs are water insoluble, they must be packagedin special molecular complexes known as “lipoproteins” in order to betransported in the plasma. Lipoproteins can accumulate in the plasma dueto overproduction and/or deficient removal. There are at least fivedistinct lipoproteins differing in size, composition, density, andfunction. In the cells of the small of the intestine, dietary lipids arepackaged into large lipoprotein complexes called “chylomicrons”, whichhave a high TG and low-cholesterol content. In the liver, TG andcholesterol esters are packaged and released into plasma as TG-richlipoprotein called very low density lipoprotein (“VLDL”), whose primaryfunction is the endogenous transport of TGs made in the liver orreleased by adipose tissue. Through enzymatic action, VLDL can be eitherreduced and taken up by the liver, or transformed into intermediatedensity lipoprotein (“IDL”). IDL, is in turn, either taken up by theliver, or is further modified to form the low density lipoprotein(“LDL”). LDL is either taken up and broken down by the liver, or istaken up by extrahepatic tissue. High density lipoprotein (“HDL”) helpsremove cholesterol from peripheral tissues in a process called reversecholesterol transport.

The term “dyslipidemia” refers to abnormal levels of lipoproteins inblood plasma including both depressed and/or elevated levels oflipoproteins (e.g., elevated levels of LDL, VLDL and depressed levels ofHDL).

Exemplary Primary Hyperlipidemia include, but are not limited to, thefollowing:

(1) Familial Hyperchylomicronemia, a rare genetic disorder which causesa deficiency in an enzyme, LP lipase, which breaks down fat molecules.The LP lipase deficiency can cause the accumulation of large quantitiesof fat or lipoproteins in the blood;

(2) Familial Hypercholesterolemia, a relatively common genetic disordercaused where the underlying defect is a series of mutations in the LDLreceptor gene that result in malfunctioning LDL receptors and/or absenceof the LDL receptors. This brings about ineffective clearance of LDL bythe LDL receptors resulting in elevated LDL and total cholesterol levelsin the plasma;

(3) Familial Combined Hyperlipidemia, also known as multiplelipoprotein-type hyperlipidemia; an inherited disorder where patientsand their affected first-degree relatives can at various times manifesthigh cholesterol and high triglycerides. Levels of HDL cholesterol areoften moderately decreased;

(4) Familial Defective Apolipoprotein B-100 is a relatively commonautosomal dominant genetic abnormality. The defect is caused by a singlenucleotide mutation that produces a substitution of glutamine forarginine which can cause reduced affinity of LDL particles for the LDLreceptor. Consequently, this can cause high plasma LDL and totalcholesterol levels;

(5) Familial Dysbetaliproteinemia, also referred to as Type IIIHyperlipoproteinemia, is an uncommon inherited disorder resulting inmoderate to severe elevations of serum TG and cholesterol levels withabnormal apolipoprotein E function. HDL levels are usually normal; and

(6) Familial Hypertriglyceridemia, is a common inherited disorder inwhich the concentration of plasma VLDL is elevated. This can cause mildto moderately elevated triglyceride levels (and usually not cholesterollevels) and can often be associated with low plasma HDL levels.

Risk factors in exemplary Secondary Hyperlipidemia include, but are notlimited to, the following: (1) disease risk factors, such as a historyof Type 1 diabetes, Type 2 diabetes, Cushing's syndrome, hypothyroidismand certain types of renal failure; (2) drug risk factors, whichinclude, birth control pills; hormones, such as estrogen, andcorticosteroids; certain diuretics; and various β blockers; (3) dietaryrisk factors include dietary fat intake per total calories greater than40%; saturated fat intake per total calories greater than 10%;cholesterol intake greater than 300 mg per day; habitual and excessivealcohol use; and obesity.

The terms “obese” and “obesity” refers to, according to the World HealthOrganization, a Body Mass Index (BMI) greater than 27.8 kg/m² for menand 27.3 kg/m² for women (BMI equals weight (kg)/height (m²). Obesity islinked to a variety of medical conditions including diabetes andhyperlipidemia. Obesity is also a known risk factor for the developmentof Type 2 diabetes (See, e.g., Barrett-Conner, E., Epidemol. Rev. (1989)11: 172-181; and Knowler, et al., Am. J. Clin. Nutr. (1991)53:1543-1551).

“Pharmaceutically acceptable salts” refer to the non-toxic alkali metal,alkaline earth metal, and ammonium salts commonly used in thepharmaceutical industry including the sodium, potassium, lithium,calcium, magnesium, barium, ammonium, and protamine zinc salts, whichare prepared by methods well known in the art. The term also includesnon-toxic acid addition salts, which are generally prepared by reactingthe compounds of the present invention with a suitable organic orinorganic acid. Representative salts include, but are not limited to,the hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate,valerate, oleate, laurate, borate, benzoate, lactate, phosphate,tosylate, citrate, maleate, fumarate, succinate, tartrate, napsylate,and the like.

“Pharmaceutically acceptable acid addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freebases and which are not biologically or otherwise undesirable, formedwith inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid and the like, and organicacids such as acetic acid, propionic acid, glycolic acid, pyruvic acid,oxalic acid, malic acid, malonic acid, succinic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid,mandelic acid, menthanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid and the like. For a descriptionof pharmaceutically acceptable acid addition salts as prodrugs. See,e.g., Bundgaard, H., ed., Design of Prodrugs (Elsevier SciencePublishers, Amsterdam 1985).

“Pharmaceutically acceptable ester” refers to those esters which retain,upon hydrolysis of the ester bond, the biological effectiveness andproperties of the carboxylic acid or alcohol and are not biologically orotherwise undesirable. For a description of pharmaceutically acceptableesters as prodrugs, see Bundgaard, H., supra. These esters are typicallyformed from the corresponding carboxylic acid and an alcohol. Generally,ester formation can be accomplished via conventional synthetictechniques. (See, e.g., March Advanced Organic Chemistry, 3rd Ed., p.1157 (John Wiley & Sons, New York 1985) and references cited therein,and Mark et al., Encyclopedia of Chemical Technology, (1980) John Wiley& Sons, New York). The alcohol component of the ester will generallycomprise: (i) a C₂-C₁₂ aliphatic alcohol that can or can not contain oneor more double bonds and can or can not contain branched carbons; or(ii) a C₇-C₁₂ aromatic or heteroaromatic alcohols. The present inventionalso contemplates the use of those compositions which are both esters asdescribed herein and at the same time are the pharmaceuticallyacceptable acid addition salts thereof.

“Pharmaceutically acceptable amide” refers to those amides which retain,upon hydrolysis of the amide bond, the biological effectiveness andproperties of the carboxylic acid or amine and are not biologically orotherwise undesirable. For a description of pharmaceutically acceptableamides as prodrugs, see, Bundgaard, H., ed., supra. These amides aretypically formed from the corresponding carboxylic acid and an amine.Generally, amide formation can be accomplished via conventionalsynthetic techniques. See, e.g., March et al., Advanced OrganicChemistry, 3rd Ed., p. 1152 (John Wiley & Sons, New York 1985), and Market al., Encyclopedia of Chemical Technology, (John Wiley & Sons, NewYork 1980). The present invention also contemplates the use of thosecompositions which are both amides as described herein and at the sametime are the pharmaceutically acceptable acid addition salts thereof.

The term “IC₅₀” refers to the concentration of a compound which wouldprovide 50% of a maximal inhibitory effect of the compound on a subjectenzyme (e.g., COX-1, cytochrome P450 2C9) under suitable assayconditions modeling the inhibitory action of the compound on the enzymeunder physiological conditions. In a preferred embodiment, the IC₅₀ forCOX-1 inhibitory activity is determined according to the method ofExample 19. In a preferred embodiment, the IC₅₀ for inhibition ofcytochrome P450 2C9 is determined according to the method of Example 7.

DETAILED DESCRIPTION (1) General

The present invention is directed to use of a preferred(−)(3-trihalomethylphenoxy)(4-halophenyl)acetic acid derivatives havingthe following general formula:

In Formula I, R is a functional group including, but not limited to, thefollowing: hydroxy, lower aralkoxy, e.g., phenyl-lower alkoxy such asbenzyloxy, phenethyloxy; di-lower alkylamino-lower alkoxy and thenontoxic, pharmacologically acceptable acid addition salts thereof,e.g., dimethylaminoethoxy, diethylaminoethoxy hydrochloride,diethylaminoethoxy citrate, diethylaminopropoxy; lower alkanamido loweralkoxy, e.g., formamidoethoxy, acetamidoethoxy or acetamidopropoxy;benzamido-lower alkoxy, e.g., benzamidoethoxy or benzamidopropoxy;ureido-lower alkoxy, e.g., ureidoethoxy or 1-methyl-2-ureidoethoxy;N′-lower alkyl-ureido-lower alkoxy, i.e., R¹NH—CONH—C_(n)H_(2n)—O—wherein R¹ represents lower alkyl and n is an integer having a value offrom 1 to about 5, e.g., N′-ethyl-ureidoethoxy orN′-ethyl-ureidopropoxy; carbamoyl-lower alkoxy, e.g., carbamoylmethoxyor carbamoylethoxy; halophenoxy substituted lower alkoxy, e.g.,2-(4-chlorophenoxy)ethoxy or 2-(4-chlorophenoxy)-2-methylpropoxy;carbamoyl substituted phenoxy, e.g., 2-carbamoylphenoxy; carboxy-loweralkylamino and the nontoxic, pharmacologically acceptable amine additionsalts thereof, e.g., carboxymethylamino cyclohexylamine salt orcarboxyethylamine; N,N-di-lower alkylamino-lower alkylamino and thenontoxic, pharmacologically acceptable acid solution salts thereof,e.g., N,N-dimethylaminoethylamino hydrochloride,N,N-diethylaminoethylamino, N,N-diethylaminoethylamino citrate, orN,N-dimethylaminopropylamino citrate; halo substituted lower alkylamino,e.g., 2-chloroethylamino or 4-chlorobutylamino; hydroxy substitutedlower alkylamino, e.g., 2-hydroxyethylamino, or 3-hydroxypropylamino;lower alkanoyloxy substituted lower alkylamino, e.g., acetoxyethylaminoor acetoxypropylamino; ureido; lower alkoxycarbonylamino, e.g.,methoxycarbonylamino (i.e., —NHCOOCH₃), or ethyoxycarbonylamino (i.e.,CHCOOC₂H₅). In a preferred embodiment, R is selected such that it is ahydrolyzable moiety, such as an ester or amide, and upon hydrolysis ofthe ester or amide bond, the compound is biologically active such aspharmaceutically acceptable esters or amides as prodrugs. X, in formulaI, is a halogen, e.g., chloro, bromo, fluoro or iodo.

In a preferred embodiment, the present invention relates to use of the(−)(3-trihalomethylphenoxy)(4-halophenyl)acetic acid derivatives havingthe following general formula:

In Formula II, R² is a functional group including, but not limited to,the following: hydrogen, phenyl-lower alkyl, e.g., benzyl; loweralkanamido-lower alkyl, e.g., acetamidoethyl; or benzamido-lower alkyl,e.g., benzamidoethyl. X, in Formula II, is a halogen, e.g., chloro,bromo, fluoro or iodo.

In a further preferred embodiment, the present invention relates to theuse of a compound having the formula:

The compound of Formula III is referred to as “(−)2-acetamidoethyl4-chlorophenyl-(3-trifluoromethylphenoxy)acetate” (also referred to as“(−) halofenate”).

Changes in drug metabolism mediated by inhibition of cytochrome P450enzymes have a very high potential to precipitate significant adverseeffects in patients. Such effects were previously noted in patientstreated with racemic halofenate. In the present studies, racemichalofenic acid was found to inhibit cytochrome P450 2C9, an enzyme knownto play a significant role in the metabolism of specific drugs. This canlead to significant problems with drug interactions with anticoagulants,anti-inflammatory agents and other drugs metabolized by this enzyme.However, quite surprisingly, a substantial difference was observedbetween the enantiomers of halofenic acid in their inability to inhibitcytochrome P450 2C9, the (−) enantiomer being about twenty-fold lessactive whereas the (+) enantiomer was quite potent (see Example 7).Thus, use of the (−) enantiomer of compounds in Formula I, Formula II orFormula III will avoid the inhibition of this enzyme and the adverseeffects on drug metabolism previously observed with racemic halofenate.

The present invention encompasses a method of modulating insulinresistance in a mammal, the method comprising: administering to themammal a therapeutically effective amount of a compound having thegeneral structure of Formula I or a pharmaceutically acceptable saltthereof. In a presently preferred embodiment, the compound has thegeneral structure of Formula II. In a further preferred embodiment, thecompound has the structure of Formula III. Quite surprisingly, themethod avoids the adverse effects associated with the administration ofa racemic mixture of halofenate by providing an amount of the (−)stereoisomer of the compounds in Formula I, Formula II or Formula IIIwhich is insufficient to cause the adverse effects associated with theinhibition of cytochrome P450 2C9.

The present invention also encompasses a method of modulating Type 2diabetes in a mammal, the method comprising: administering to the mammala therapeutically effective amount of a compound having the generalstructure of Formula I or a pharmaceutically acceptable salt thereof. Ina presently preferred embodiment, the compound has the general structureof Formula II. In a further preferred embodiment, the compound has thestructure of Formula III. Quite surprisingly, the method avoids theadverse effects associated with the administration of a racemic mixtureof halofenate by providing an amount of the (−) stereoisomer of thecompounds in Formula I, Formula II or Formula III which is insufficientto cause the adverse effects associated with the inhibition ofcytochrome P450 2C9.

The present invention further encompasses a method of modulatinghyperlipidemia in a mammal, the method comprising: administering to themammal a therapeutically effective amount of a compound having thegeneral structure of Formula I or a pharmaceutically acceptable saltthereof. In a presently preferred embodiment, the compound has thegeneral structure of Formula II. In a further preferred embodiment, thecompound has the structure of Formula III. Quite surprisingly, themethod avoids the adverse effects associated with the administration ofa racemic mixture of halofenate by providing an amount of the (−)stereoisomer of the compounds in Formula I, Formula II or Formula IIIwhich is insufficient to cause the adverse effects associated with theinhibition of cytochrome P450 2C9.

The racemic mixture of the halofenate (i.e., a 1:1 racemic mixture ofthe two enantiomers) possesses antihyperlipidemic activity and providestherapy and a reduction of hyperglycemia related to diabetes whencombined with certain other drugs commonly used to treat this disease.However, this racemic mixture, while offering the expectation ofefficacy, causes adverse effects. The term “adverse effects” includes,but is not limited to, nausea, gastrointestinal ulcers, andgastrointestinal bleeding. Other side effects that have been reportedwith racemic halofenate include potential problems with drug-druginteractions, especially including difficulties controllinganticoagulation with Coumadin™. Utilizing the substantially purecompounds of the present invention results in clearer dose relateddefinitions of efficacy, diminished adverse effects, and accordingly, animproved therapeutic index. As such, it has now been discovered that itis more desirable and advantageous to administer the (−) enantiomer ofhalofenate instead of racemic halofenate.

The present invention further encompasses a method of modulatinghyperuricemia in a mammal, the method comprising: administering to themammal a therapeutically effective amount of a compound having thegeneral structure of Formula I or a pharmaceutically acceptable saltthereof. In a presently preferred embodiment, the compound has thegeneral structure of Formula II. In a further preferred embodiment, thecompound has the structure of Formula III. Quite surprisingly, themethod avoids the adverse effects associated with the administration ofa racemic mixture of halofenate by providing an amount of the (−)stereoisomer of the compounds in Formula I, Formula II or Formula IIIwhich is insufficient to cause the adverse effects associated with theinhibition of cytochrome P450 2C9.

(2) (−) Enantiomers of Formula I, Formula II and Formula III

Many organic compounds exist in optically active forms, i.e., they havethe ability to rotate the plane of plane-polarized light. In describingan optically active compound, the prefixes R and S are used to denotethe absolute configuration of the molecule about its chiral center(s).The prefixes “d” and “l” or (+) and (−) are employed to designate thesign of rotation of plane-polarized light by the compound, with (−) or lmeaning that the compound is “levorotatory” and with (+) or d is meaningthat the compound is “dextrorotatory”. There is no correlation betweennomenclature for the absolute stereochemistry and for the rotation of anenantiomer. For a given chemical structure, these compounds, called“stereoisomers,” are identical except that they are mirror images of oneanother. A specific stereoisomer can also be referred to as an“enantiomer,” and a mixture of such isomers is often called an“enantiomeric” or “racemic” mixture. See, e.g., Streitwiesser, A. &Heathcock, C. H., INTRODUCTION TO ORGANIC CHEMISTRY, 2^(nd) Edition,Chapter 7 (MacMillan Publishing Co., U.S.A. 1981).

The chemical synthesis of the racemic mixture of halofenates(3-trihalomethylphenoxy)(4-halophenyl)acetic acid derivatives can beperformed by the methods described in U.S. Pat. No. 3,517,050, theteaching of which are incorporated herein by reference. The synthesis ofthe compounds of the present invention is further described in theExamples, supra. The individual enantiomers can be obtained byresolution of the racemic mixture of enantiomers using conventionalmeans known to and used by those of skill in the art. See, e.g., Jaques,J., et al., in ENANTIOMERS, RACEMATES, AND RESOLUTIONS, John Wiley andSons, New York (1981). Other standard methods of resolution known tothose skilled in the art, including but not limited to, simplecrystallization and chromatographic resolution, can also be used (see,e.g., STEREOCHEMISTRY OF CARBON COMPOUNDS (1962) E. L. Eliel, McGrawHill; Lochmuller, J. Chromatography (1975) 113, 283-302). Additionally,the compounds of the present invention, i.e., the optically pureisomers, can be prepared from the racemic mixture by enzymaticbiocatalytic resolution. Enzymatic biocatalytic resolution has beendescribed previously (see, e.g., U.S. Pat. Nos. 5,057,427 and 5,077,217,the disclosures of which are incorporated herein by reference). Othermethods of obtaining enantiomers include stereospecific synthesis (see,e.g., Li, A. J. et al., Pharm. Sci. (1997) 86: 1073-1077).

The term “substantially free of its (+) stereoisomer,” as used herein,means that the compositions contain a substantially greater proportionof the (−) isomer of halofenate in relation to the (+) isomer. In apreferred embodiment, the term “substantially free of its (+)stereoisomer,” as used herein, means that the composition is at least90% by weight of the (−) isomer and 10% by weight or less of the (+)isomer. In a more preferred embodiment, the term “substantially free ofits (+) stereoisomer,” as used herein, means that the compositioncontains at least 99% by weight of the (−) isomer and 1% by weight orless of the (+) isomer. In the most preferred embodiment, the term“substantially free of its (+) stereoisomer,” means that the compositioncontains greater than 99% by weight of the (−) isomer. These percentagesare based upon the total amount of halofenate in the composition. Theterms “substantially optically pure (l) isomer of halofenate,”“substantially optically pure (l) halofenate,” “optically pure (l)isomer of halofenate” and “optically pure (l) halofenate” all refer tothe (−) isomer and are encompassed by the above-described amounts. Inaddition, the terms “substantially optically pure (d) isomer ofhalofenate,” “substantially optically pure (d) halofenate,” “opticallypure (d) isomer of halofenate” and “optically pure (d) halofenate” allrefer to the (+) isomer and are encompassed by the above-describedamounts.

The term “enantiomeric excess” or “ee” is related to the term “opticalpurity” in that both are measures of the same phenomenon. The value ofee will be a number from 0 to 100, 0 being racemic and 100 being pure,single enantiomer. A compound that is referred to as 98% optically purecan be described as 96% ee.

(3) Combination Therapy with Additional Active Agents

The compositions can be formulated and administered in the same manneras detailed below. “Formulation” is defined as a pharmaceuticalpreparation that contains a mixture of various excipients and keyingredients that provide a relatively stable, desirable and useful formof a compound or drug. For the present invention, “formulation” isincluded within the meaning of the term “composition.” The compounds ofthe present invention can be used effectively alone or in combinationwith one or more additional active agents depending on the desiredtarget therapy (see, e.g., Turner, N. et al., Prog. Drug Res. (1998) 51:33-94; Haffner, S. Diabetes Care (1998) 21: 160-178; and DeFronzo, R. etal. (eds.), Diabetes Reviews (1997) Vol. 5 No. 4). A number of studieshave investigated the benefits of combination therapies with oral agents(see, e.g., Mahler, R., J. Clin. Endocrinol. Metab. (1999) 84: 1165-71;United Kingdom Prospective Diabetes Study Group: UKPDS 28, Diabetes Care(1998) 21: 87-92; Bardin, C. W., (ed.), CURRENT THERAPY IN ENDOCRINOLOGYAND METABOLISM, 6^(th) Edition (Mosby—Year Book, Inc., St. Louis, Mo.1997); Chiasson, J. et al., Ann. Intern. Med. (1994) 121: 928-935;Coniff, R. et al., Clin. Ther. (1997) 19: 16-26; Coniff, R. et al., Am.J. Med. (1995) 98: 443-451; and Iwamoto, Y. et al., Diabet. Med. (1996)13 365-370; Kwiterovich, P. Am. J. Cardiol (1998) 82(12A): 3U-17U).These studies indicate that diabetes and hyperlipidemia modulation canbe further improved by the addition of a second agent to the therapeuticregimen. Combination therapy includes administration of a singlepharmaceutical dosage formulation which contains a compound having thegeneral structure of Formula I (or Formula II or Formula III) and one ormore additional active agents, as well as administration of a compoundof Formula I (or Formula II or Formula III) and each active agent in itsown separate pharmaceutical dosage formulation. For example, a compoundof Formula I and an HMG-CoA reductase inhibitor can be administered tothe human subject together in a single oral dosage composition, such asa tablet or capsule, or each agent can be administered in separate oraldosage formulations. Where separate dosage formulations are used, acompound of Formula I and one or more additional active agents can beadministered at essentially the same time (i.e., concurrently), or atseparately staggered times (i.e., sequentially). Combination therapy isunderstood to include all these regimens.

An example of combination therapy that modulates (prevents the onset ofthe symptoms or complications associated) atherosclerosis, wherein acompound of Formula I is administered in combination with one or more ofthe following active agents: an antihyperlipidemic agent; a plasmaHDL-raising agent; an antihypercholesterolemic agent, such as acholesterol biosynthesis inhibitor, e.g., an hydroxymethylglutaryl (HMG)CoA reductase inhibitor (also referred to as statins, such aslovastatin, simvastatin, pravastatin, fluvastatin, and atorvastatin), anHMG-CoA synthase inhibitor, a squalene epoxidase inhibitor, or asqualene synthetase inhibitor (also known as squalene synthaseinhibitor); an acyl-coenzyme A cholesterol acyltransferase (ACAT)inhibitor, such as melinamide; probucol; nicotinic acid and the saltsthereof and niacinamide; a cholesterol absorption inhibitor, such asβ-sitosterol; a bile acid sequestrant anion exchange resin, such ascholestyramine, colestipol or dialkylaminoalkyl derivatives of across-linked dextran; an LDL (low density lipoprotein) receptor inducer;fibrates, such as clofibrate, bezafibrate, fenofibrate, and gemfibrizol;vitamin B₆ (also known as pyridoxine) and the pharmaceuticallyacceptable salts thereof, such as the HCl salt; vitamin B₁₂ (also knownas cyanocobalamin); vitamin B₃ (also known as nicotinic acid andniacinamide, supra); anti-oxidant vitamins, such as vitamin C and E andbeta carotene; a beta-blocker; an angiotensin II antagonist; anangiotensin converting enzyme inhibitor; and a platelet aggregationinhibitor, such as fibrinogen receptor antagonists (i.e., glycoproteinIIb/IIIa fibrinogen receptor antagonists) and aspirin. As noted above,the compounds of Formula I can be administered in combination with morethan one additional active agent, for example, a combination of acompound of Formula I with an HMG-CoA reductase inhibitor (e.g.,lovastatin, simvastatin and pravastatin) and aspirin, or a compound ofFormula I with an HMG-CoA reductase inhibitor and β blocker.

Another example of combination therapy can be seen in treating obesityor obesity-related disorders, wherein the compounds of Formula I can beeffectively used in combination with, for example, phenylpropanolamine,phentermine, diethylpropion, mazindol; fenfluramine, dexfenfluramine,phentiramine, β₃ adrenoceptor agonist agents; sibutramine,gastrointestinal lipase inhibitors (such as orlistat), and leptins.Other agents used in treating obesity or obesity-related disorderswherein the compounds of Formula I can be effectively used incombination with, for example, neuropeptide Y, enterostatin,cholecytokinin, bombesin, amylin, histamine H₃ receptors, dopamine D₂receptors, melanocyte stimulating hormone, corticotrophin releasingfactor, galanin and gamma amino butyric acid (GABA).

Still another example of combination therapy can be seen in modulatingdiabetes (or treating diabetes and its related symptoms, complications,and disorders), wherein the compounds of Formula I can be effectivelyused in combination with, for example, sulfonylureas (such aschlorpropamide, tolbutamide, acetohexamide, tolazamide, glyburide,gliclazide, glynase, glimepiride, and glipizide), biguanides (such asmetformin), thiazolidinediones (such as ciglitazone, pioglitazone,troglitazone, and rosiglitazone); dehydroepiandrosterone (also referredto as DHEA or its conjugated sulphate ester, DHEA-SO₄);antiglucocorticoids; TNFα inhibitors; α-glucosidase inhibitors (such asacarbose, miglitol, and voglibose), pramlintide (a synthetic analog ofthe human hormone amylin), other insulin secretogogues (such asrepaglinide, gliquidone, and nateglinide), insulin, as well as theactive agents discussed above for treating atherosclerosis.

A further example of combination therapy can be seen in modulatinghyperlipidemia (treating hyperlipidemia and its related complications),wherein the compounds of Formula I can be effectively used incombination with, for example, statins (such as fluvastatin, lovastatin,pravastatin or simvastatin), bile acid-binding resins (such ascolestipol or cholestyramine), nicotinic acid, probucol, betacarotene,vitamin E, or vitamin C.

In accordance with the present invention, a therapeutically effectiveamount of a compound of Formula I (or Formula II or Formula III) can beused for the preparation of a pharmaceutical composition useful fortreating diabetes, treating hyperlipidemia, treating hyperuricemia,treating obesity, lowering triglyceride levels, lowering cholesterollevels, raising the plasma level of high density lipoprotein, and fortreating, preventing or reducing the risk of developing atherosclerosis.

Additionally, an effective amount of a compound of Formula I (or FormulaII or Formula III) and a therapeutically effective amount of one or moreactive agents selected from the group consisting of: anantihyperlipidemic agent; a plasma HDL-raising agent; anantihypercholesterolemic agent, such as a cholesterol biosynthesisinhibitor, for example, an HMG-CoA reductase inhibitor, an HMG-CoAsynthase inhibitor, a squalene epoxidase inhibitor, or a squalenesynthetase inhibitor (also known as squalene synthase inhibitor); anacyl-coenzyme A cholesterol acyltransferase inhibitor; probucol;nicotinic acid and the salts thereof; niacinamide; a cholesterolabsorption inhibitor; a bile acid sequestrant anion exchange resin; alow density lipoprotein receptor inducer; clofibrate, fenofibrate, andgemfibrozil; vitamin B₆ and the pharmaceutically acceptable saltsthereof; vitamin B₁₂; an anti-oxidant vitamin; β-blocker; an angiotensinII antagonist; an angiotensin converting enzyme inhibitor; a plateletaggregation inhibitor; a fibrinogen receptor antagonist; aspirin;phentiramines, β₃ adrenergic receptor agonists; sulfonylureas,biguanides, α-glucosidase inhibitors, other insulin secretogogues, andinsulin can be used together for the preparation of a pharmaceuticalcomposition useful for the above-described treatments.

(4) Pharmaceutical Formulations and Methods of Administration

In the methods of the present invention, the compounds of Formula I,Formula II, and Formula III can be delivered or administered to amammal, e.g., a human patient or subject, alone, in the form of apharmaceutically acceptable salt or hydrolysable precursor thereof, orin the form of a pharmaceutical composition where the compound is mixedwith suitable carriers or excipient(s) in a therapeutically effectiveamount. By a “therapeutically effective dose”, “therapeuticallyeffective amount”, or, interchangeably, “pharmacologically acceptabledose” or “pharmacologically acceptable amount”, it is meant that asufficient amount of the compound of the present invention,alternatively, a combination, for example, a compound of the presentinvention, which is substantially free of its (+) stereoisomer, and apharmaceutically acceptable carrier, will be present in order to achievea desired result, e.g., alleviating a symptom or complication of Type 2diabetes.

The compounds of Formula I, Formula II, and Formula III that are used inthe methods of the present invention can be incorporated into a varietyof formulations for therapeutic administration. More particularly, thecompounds of Formula I (or Formula II or Formula III) can be formulatedinto pharmaceutical compositions by combination with appropriate,pharmaceutically acceptable carriers or diluents, and can be formulatedinto preparations in solid, semi-solid, liquid or gaseous forms, such astablets, capsules, pills, powders, granules, dragees, gels, slurries,ointments, solutions, suppositories, injections, inhalants and aerosols.As such, administration of the compounds can be achieved in variousways, including oral, buccal, rectal, parenteral, intraperitoneal,intradermal, transdermal, intratracheal administration. Moreover, thecompound can be administered in a local rather than systemic manner, ina depot or sustained release formulation. In addition, the compounds canbe administered in a liposome.

In addition, the compounds of Formula I, Formula II or Formula III canbe formulated with common excipients, diluents or carriers, andcompressed into tablets, or formulated as elixirs or solutions forconvenient oral administration, or administered by the intramuscular orintravenous routes. The compounds can be administered transdermally, andcan be formulated as sustained release dosage forms and the like.

Compounds of Formula I, Formula II, or Formula III can be administeredalone, in combination with each other, or they can be used incombination with other known compounds (discussed supra). Inpharmaceutical dosage forms, the compounds can be administered in theform of their pharmaceutically acceptable salts thereof. They cancontain hydrolyzable moieties. They can also be used alone or inappropriate association, as well as in combination with, otherpharmaceutically active compounds.

Suitable formulations for use in the present invention are found inRemington's Pharmaceutical Sciences (Mack Publishing Company (1985)Philadelphia, Pa., 17th ed.), which is incorporated herein by reference.Moreover, for a brief review of methods for drug delivery, see, Langer,Science (1990) 249:1527-1533, which is incorporated herein by reference.The pharmaceutical compositions described herein can be manufactured ina manner that is known to those of skill in the art, i.e., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes. Thefollowing methods and excipients are merely exemplary and are in no waylimiting.

For injection, the compounds can be formulated into preparations bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives. Preferably, the compounds of the present invention can beformulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiological saline buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the compounds of Formula I, Formula II, orFormula III can be formulated readily by combining with pharmaceuticallyacceptable carriers that are well known in the art. Such carriers enablethe compounds to be formulated as tablets, pills, dragees, capsules,emulsions, lipophilic and hydrophilic suspensions, liquids, gels,syrups, slurries, suspensions and the like, for oral ingestion by apatient to be treated. Pharmaceutical preparations for oral use can beobtained by mixing the compounds with a solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are, in particular, fillers such assugars, including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents can beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions can be used, which can optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments can be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds can be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers can be added. All formulations fororal administration should be in dosages suitable for suchadministration.

In a preferred embodiment, the preparations are enteric coated to reduceexposure of the stomach to the active agent.

For buccal administration, the compositions can take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas, or from propellant-free, dry-powder inhalers. In thecase of a pressurized aerosol the dosage unit can be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insufflator can be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compounds can be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection can be presented in unit dosage form, e.g., in ampules orin multidose containers, with an added preservative. The compositionscan take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and can contain formulator agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds can be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions can contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension can also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.Alternatively, the active ingredient can be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds can also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter, carbowaxes, polyethylene glycolsor other glycerides, all of which melt at body temperature, yet aresolidified at room temperature.

In addition to the formulations described previously, the compounds canalso be formulated as a depot preparation. Such long acting formulationscan be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds can be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

Alternatively, other delivery systems for hydrophobic pharmaceuticalcompounds can be employed. Liposomes and emulsions are well knownexamples of delivery vehicles or carriers for hydrophobic drugs. In apresently preferred embodiment, long-circulating, i.e., stealth,liposomes can be employed. Such liposomes are generally described inWoodle, et al., U.S. Pat. No. 5,013,556, the teaching of which is herebyincorporated by reference. The compounds of the present invention canalso be administered by controlled release means and/or delivery devicessuch as those described in U.S. Pat. Nos. 3,845,770; 3,916,899;3,536,809; 3,598,123; and 4,008,719; the disclosures of which are herebyincorporated by reference.

Certain organic solvents such as dimethylsulfoxide (DMSO) also can beemployed, although usually at the cost of greater toxicity.Additionally, the compounds can be delivered using a sustained-releasesystem, such as semipermeable matrices of solid hydrophobic polymerscontaining the therapeutic agent. Various types of sustained-releasematerials have been established and are well known by those skilled inthe art. Sustained-release capsules can, depending on their chemicalnature, release the compounds for a few hours up to over 100 days.

The pharmaceutical compositions also can comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in atherapeutically effective amount. The amount of composition administeredwill, of course, be dependent on the subject being treated, on thesubject's weight, the severity of the affliction, the manner ofadministration and the judgment of the prescribing physician.Determination of an effective amount is well within the capability ofthose skilled in the art, especially in light of the detailed disclosureprovided herein.

For any compound used in the method of the present invention, atherapeutically effective dose can be estimated initially from cellculture assays or animal models.

Moreover, toxicity and therapeutic efficacy of the compounds describedherein can be determined by standard pharmaceutical procedures in cellcultures or experimental animals, e.g., by determining the LD₅₀, (thedose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). Compounds can bescreened for their ability to inhibit cytochrome P450 c29 by the methodsof example 7. Such methods are well known to one of ordinary skill inthe art. The compounds of the present invention can be screened fortheir ability to inhibit the COX-1 enzyme by any such enzyme assay aswould be well known to one of ordinary skill in the art. In particular,the methods of example 19 are exemplary for measuring COX-1 enzymeinhibition.

The dose ratio between toxic and therapeutic effect is the therapeuticindex and can be expressed as the ratio between LD₅₀ or TD₅₀ and ED₅₀.Compounds which exhibit high therapeutic indices are preferred. In vitroIC₅₀ data can be used to select promising compounds for in vivo toxicityand efficacy dose response studies. The data obtained from these cellculture assays and animal studies can be used in formulating a dosagerange and compounds that are efficacious and not toxic for use in human.The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage can vary within this range depending upon thedosage form employed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. (See, e.g.,Fingl et al., 1975 In: The Pharmacological Basis of Therapeutics, Ch.1). In a preferred embodiment the compounds for use according to theinvention are the (−) stereoisomers of compounds of Formula I, II, orIII which have a therapeutic index which is at least 4-fold greater thanthat of the corresponding (+) stereoisomer with respect to theinhibition of cytochrome P450 2C9 or COX-1.

The amount of active compound that can be combined with a carriermaterial to produce a single dosage form will vary depending upon thedisease treated, the mammalian species, and the particular mode ofadministration. However, as a general guide, suitable unit doses for thecompounds of the present invention can, for example, preferably containbetween 100 mg to about 3000 mg of the active compound. Preferred unitdoses are from about 50 to 100 mg, 100 to 250 mg, 100 to 500 mg, andfrom about 500 to about 1000 mg. Such unit doses can be administeredmore than once a day, for example 2, 3, 4, 5 or 6 times a day, butpreferably 1 or 2 times per day, so that the total daily dosage for a 70kg adult is in the range of 0.1 to about 250 mg per kg weight ofsubject. A preferred daily dosage is 5 to about 25 mg per kg weight ofsubject, and such therapy can extend for a number of weeks or months,and in some cases, years. In other embodiments, the daily dosage wouldbe from 1 to 5 mg per kg of body weight or 5 to 25 mg per kg of bodyweight. It will be understood, however, that the specific dose level forany particular patient will depend on a variety of factors including theactivity of the specific compound employed; the age, body weight,general health, sex and diet of the individual being treated; the timeand route of administration; the rate of excretion; other drugs whichhave previously been administered; and the severity of the particulardisease undergoing therapy, as is well understood by those of skill inthe area.

A typical dosage can be one 10 to about 1500 mg tablet taken once a day,or, multiple times per day, or one time-release capsule or tablet takenonce a day and containing a proportionally higher content of activeingredient. The time-release effect can be obtained by capsule materialsthat dissolve at different pH values, by capsules that release slowly byosmotic pressure, or by any other known means of controlled release.

It can be necessary to use dosages outside these ranges in some cases aswill be apparent to those skilled in the art. Further, it is noted thatthe clinician or treating physician will know how and when to interrupt,adjust, or terminate therapy in conjunction with individual patientresponse.

In some embodiments, the active compound of Formula I, II, or III isadministered in an amount which provides a blood or plasma concentrationwhich is below the IC₅₀ for inhibition of the COX-1 enzyme in blood bythe compound. In some embodiments, the active compound is the (−) isomerand is administered in an amount and by a route which provides no morethan a 5%, 10%, 20%, or 40% inhibition of the plasma COX-1 enzyme at anytime after administration or no more than a 5%, 10%, 20%, or 40%inhibition of the plasma COX-1 enzyme as an average over the period oftime between a repeated dosage. In a preferred embodiment, the route ofadministration is oral. In other embodiments, the subject is human. Insome embodiments, the compound is (−) halofenate or a prodrug of (−)halofenic acid. In some embodiments, the inhibition of the COX-1 enzymeis determined by the methodology of Example 19 below.

(5) Protecting Groups

Certain compounds having the general structure of Formula I and II mayrequire the use of protecting groups to enable their successfulelaboration into the desired structure. Protecting groups can be chosenwith reference to Greene, T. W., et al., Protective Groups in OrganicSynthesis, John Wiley & Sons, Inc., 1991. The blocking groups arereadily removable, i.e., they can be removed, if desired, by procedureswhich will not cause cleavage or other disruption of the remainingportions of the molecule. Such procedures include chemical and enzymatichydrolysis, treatment with chemical reducing or oxidizing agents undermild conditions, treatment with fluoride ion, treatment with atransition metal catalyst and a nucleophile, and catalytichydrogenation.

Examples of suitable hydroxyl protecting groups are: trimethylsilyl,triethylsilyl, o-nitrobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,t-butyldiphenylsilyl, t-butyldimethylsilyl, benzyloxycarbonyl,t-butyloxycarbonyl, 2,2,2-trichloroethyloxycarbonyl, andallyloxycarbonyl. Examples of suitable carboxyl protecting groups arebenzhydryl, o-nitrobenzyl, p-nitrobenzyl, 2-naphthylmethyl, allyl,2-chloroallyl, benzyl, 2,2,2-trichloroethyl, trimethylsilyl,t-butyldimethylsilyl, t-butyldiphenylsilyl, 2-(trimethylsilyl)ethyl,phenacyl, p-methoxybenzyl, acetonyl, p-methoxyphenyl, 4-pyridylmethyland t-butyl.

(6) Process

Processes for making the compounds of the present invention aregenerally depicted in Schemes 1 and 2 (and further described in theExamples):

According to Scheme 1, a substituted phenyl acetonitrile is converted toa substituted phenyl acetic acid. The substituted phenyl acetic acid isconverted to an activated acid derivative (e.g., acid chloride),followed by halogenation at the alpha-carbon and esterification with analcohol. The halogenated ester is treated with a substituted phenol(e.g., 3-trifluoromethylphenol), yielding an aryl ether, which ishydrolyzed to form a carboxylic acid derivative. The acid derivated isconverted to an activated acid derivative and subsequently treated witha nucleophile (e.g., N-acetylethanolamine) to afford the desiredproduct.

According to Scheme 2, a substituted phenyl acetic acid is converted toan activated acid derivative (e.g., acid chloride) followed byhalogenation at the alpha-carbon. The activated acid portion of themolecule is reacted with a nucleophile (e.g., N-acetylethanolamine) toprovide a protected acid. The halogenated, protected acid is treatedwith a substituted phenol (e.g., 3-trifluoromethylphenol), yielding thedesired product.

The stereoisomers of the compounds of the present invention can beprepared by using reactants or reagents or catalysts in their singleenantiomeric form in the process wherever possible or by resolving themixture of stereoisomers by conventional methods, discussed supra and inthe Examples. Some of the preferred methods include use of microbialresolution, resolving the diastereomeric salts formed with chiral acidsor chiral bases and chromatography using chiral supports.

(7) Kits

In addition, the present invention provides for kits with unit doses ofthe compounds of Formula I, Formula II, or Formula III either in oral orinjectable doses. In addition to the containers containing the unitdoses will be an informational package insert describing the use andattendant benefits of the drugs in alleviating symptoms and/orcomplications associated with Type 2 diabetes as well as in alleviatinghyperlipidemia and hyperuricemia. Preferred compounds and unit doses arethose described herein above.

EXAMPLES

The compounds of Formula I, Formula II, or Formula III of the presentinvention can be readily prepared using the process set forth in Scheme1, supra, and from the following examples.

Example 1

This example relates to the preparation of MethylBromo-(4-chlorophenyl)-acetate.

The initial compound listed in Scheme 1, i.e., 4-chlorophenylaceticacid, is readily available from several commercial sources (e.g.,Aldrich and Fluka).

A 5-L Morton reactor equipped with a magnetic stirrer, a pot temperaturecontrol, and addition funnel was vented through a gas scrubber andcharged with p-chlorophenylacetic acid (720 gm, 4.2 moles) and SOCl₂(390ml, 630 gm, 5.3 moles). The reaction was stirred, heated and held at55°±5° C. for 1 hour. Bromine (220 ml., 670 gm, 5.3 moles) was thenadded over 20 min. and stirred at 55°±5° C. for 16 hours. Thetemperature was raised to 80° C. for 7 hours and then cooled to 9° C. inan ice-water bath. Methanol (2.0 L, 1.6 kg, 49.4 moles) was thencarefully added. The solvent was stripped to obtain 2 liquids weighing1.28 kg. These were dissolved in a mixture of 0.84 L water and 2.1 Lether and separated. The organic phase was washed once with 0.78 L 25%(w:w) aqueous NaCl and dried over 0.13 kg MgSO₄. This was filteredthrough Whatman #1 filter paper and stripped of solvent to obtain 0.985kg of orange liquid. The proton NMR showed this to be 80% product and19% non-brominated ester. The HPLC showed 82% product and 18%non-brominated ester. HPLC was run on a Zorbax SB-C8 column at 30° C.measuring 250×4.6 mm and 5μ particle size. The mobile phase was 60:40(v:v) acetonitrile: 0.1% H₃PO₄ at 1.5 ml/min. Detection was at 210 nm.The injected sample of 1 μl was dissolved in acetonitrile at aconcentration of 10 mg/ml. The product had a retention time of 5.0 min.and that of the non-brominated ester was 3.8 min. This crude product waspurified by vacuum distillation to obtain 96% pure product with an 84%yield. The product proton NMR (CDCl₃, 300 MHz) showed shifts at 3.79 (s,3H), 5.32 (s, 1H) and 7.20-7.55 (m, 4H) ppm.

Example 2

This example relates to the preparation of Methyl4-Chlorophenyl-(3-trifluoromethylphenoxy)-acetate.

This step was similar to the same step in U.S. Pat. No. 3,517,050 withone exception; potassium t-butoxide was used in place of sodiummethoxide to prevent generation of the corresponding methyl ether. A 5-LMorton reactor equipped with an overhead stirrer, a pot temperaturedetector, and addition funnel and under a nitrogen atmosphere wascharged with methyl bromo-(4-chlorophenyl)-acetate (830 gm, 3.0 moles)and THF (600 ml). The reactor was cooled to 14°±3° C. in an ice-waterbath and then a similarly cooled solution of trifluoromethyl-m-cresol(530 gm, 3.3 moles) in 1.0 M potassium t-butoxide in THF (3.1 L, 3.1moles) was added. The reaction proceeded exothermically with a typicaltemperature rise exceeding 25° C. and the addition was controlled tomaintain a temperature of 15°±2° C. and stirred at ambient temperaturefor 2 hours. HPLC was run on a Zorbax SB-C8 column at 30° C. measuring250×4.6 mm and 5μ particle size. The mobile phase was 60:40 (v:v)acetonitrile: 0.1% H₃PO₄ at 1.5 ml/min. Detection was at 210 nm. Theinjected sample of 1 μl was dissolved in acetonitrile at a concentrationof 10 mg/ml. The product had a retention time of 9.6 min., the startingester eluted at 5.0 min., the phenol at 3.0 and the non-brominated esterat 3.8 min. The solvent was stripped using a rotary evaporator to obtaina yellow slush that was dissolved in a mixture of 4.0 L water and 12.0 Lether. The mixture was separated and the organic phase was washed oncewith 1.6 L 5% (w:w) aqueous NaOH followed by 1.6 L water and finally 1.6L 25% (w:w) aqueous NaCl. The organic phase was dried over 0.32 kg MgSO₄and filtered through Whatman #1 filter paper. The solvent was strippedto obtain 1.0 kg of damp, off-white crystals. This was recrystallized onthe rotary evaporator by dissolving in 1.0 L methylcyclohexane at 75° C.and then cooling to 20° C. The crystals were filtered through Whatman #1filter paper and washed with three 0.25 L portions of cool (15° C.)methylcyclohexane. The wet product (0.97 kg) was dried overnight toobtain 0.81 kg of 98% pure product that corresponds to a 79% yield. Theproduct proton NMR (CDCl₃, 300 MHz) shows shifts at 3.75 (s, 3H), 5.63(s, 1H) and 7.05-7.55 (m, 8H).

Example 3

This example relates to the preparation of4-Chlorophenyl-(3-trifluoromethylphenoxy)-acetic Acid

A 12-L Morton reactor with magnetic stirrer, pot temperature controller,a reflux condenser and under a nitrogen atmosphere was charged withmethyl 4-chlorophenyl-(3-trifluoromethylphenoxy)-acetate (810 gm, 2.3moles) and absolute ethanol (5.8 L) and heated with stirring to 57° C.to dissolve the solid. A solution of KOH (520 gm, 9.3 moles) in 0.98 Lwater was added. The solution was refluxed for 30 min. and solvent wasstripped by a rotary evaporator to obtain 2.03 kg of a mixture of twonearly colorless liquids. These were dissolved in water (16 L) andtreated with 16 gm neutral Norit, then filtered through a pad ofinfusorial earth retained on Whatman #1 filter paper. The pH of thefiltrate was lowered from an initial range of 13 to a range of 1 to 2 byadding a total of 2.75 L of 3 M HCl (8.25 moles). A very sticky solidformed after the addition of the first 2.30 L of acid and ether (7 L)was added at this point. The two layers were separated and the organiclayer was dried over MgSO₄ (230 gm) and filtered through Whatman #1filter paper. The solvent was then stripped to obtain 0.85 kgwater-white syrup. The material was then recrystallized on the rotaryevaporator by adding methylcyclohexane (800 ml) and cooling to 18° C.with slow rotation. The temperature was then dropped to 5° C., thecrystals were filtered, and washed 5 times with 0.10 L portions of cold(0° C.) methylcyclohexane to obtain 0.59 kg wet crystals. The wetcrystals were dried to obtain 0.48 kg (62% yield) product with nop-chlorophenylacetic acid detectable in the proton NMR. The productproton NMR (CDCl₃, 300 MHz) shows shifts at 5.65 (s, 1H), 7.02-7.58 (m,8H) and 10.6 (s, 1H).

Example 4

This example relates to the preparation of resolved enantiomers of4-Chlorophenyl-(3-trifluoromethylphenoxy)-acetic Acid.

A 12-L open-top Morton reactor with an overhead stirrer was charged with4-chlorophenyl-(3-trifluoromethyl-phenoxy)-acetic acid (350 gm, 1.06moles) and isopropanol (4.0 L) and heated to 65°±3° C. A slurry of (−)cinchonidine (300 gm, 1.02 moles) in isopropanol (2.0 L) was added,rinsing all solid into the reactor with an additional 0.8 L ofisopropanol. The temperature dropped from 65° to 56° C. and atransparent, orange solution ultimately formed and the mixture was heldat 55°±5° C. for 2 hours. Fine crystals were collected by filtrationthrough Whatman #1 filter paper, washing once with 0.7 L hot (55° C.)isopropanol. The crystals were dried for 16 hours at ambient temperaturein a 12.6-L vacuum oven under a 5 LPM nitrogen flow. The dry solidweighed 0.37 kg and had an 80% enantiomeric excess (ee) of the (+)enantiomer. The enantiomeric excess was determined by HPLC using a250×4.6 mm R,R-WhelkO-1 column at ambient temperature. Injected sampleswere 20 μl of 2 mg/ml solutions of the samples in ethanol. The columnwas eluted with 95:5:0.4, hexane:isopropanol:acetic acid at a flow of 1ml/min. Detection was at 210 nm. The (+) enantiomer eluted at 7 to 8min. and the (−) enantiomer at 11 to 13 min. The mother liquor dropped asecond crop almost immediately that was filtered, washed, and dried toafford 0.06 kg salt that has a 90% ee of the (−)-enantiomer. Similarlythird, fourth and fifth crops weighing 0.03 kg, 0.03 kg and 0.7 kg,respectively, were obtained; with (−) enantiomer excesses of 88%, 89%and 92%, respectively.

The crude (+) salt (320 gm) was recrystallized from a mixture of ethanol(5.9 L) methanol (1.2 L). The mixture was heated with overhead stirringto dissolve, cooled at ambient temperature for 16 hours, filtered andwashed twice with 0.20 L of 5:1 (v:v) ethanol:methanol. The crystalswere dried to obtain 0.24 kg of the (+) enantiomer that had an ee of97%. This corresponded to an 80% recovery of this isomer. The resolvedsalt was suspended in a mixture of ether (6.5 L) and water (4.0 L) withoverhead stirring. The pH was lowered to 0-1 as measured by pHindicating strips with a solution of concentrated H2SO4 (0.13 L) inwater (2.5 L). The phases were separated and the organic phase andwashed twice with 6.5 L portions of water. Ether (1.9 L) was added andthe organic layer washed once more with 6.5 L water. After the finalseparation, 0.1 L of 25% (w:w) aqueous NaCl was added clean up anyslight emulsion. The product was dried over 0.19 kg MgSO₄, filtered andsolvent removed solvent to obtain 0.13 kg of water-white syrup thatsolidifies on cooling. This corresponded to a 97% recovery of productthat had a 95% ee of the (+) enantiomer. [α]_(D)+5.814° (c.=0.069 inmethyl alcohol).

The combined, crude (−) salt (200 gm) was recrystallized fromisopropanol (3.1 L). The mixture was heated to dissolve almost all ofthe solid and fast-filtered to remove insoluble solids. The mixture wasthen cooled with stirring at ambient temperature for 16 hours, filtered,washed, and dried to obtain 0.16 kg of the (−) enantiomer that has an eeof 97%. This corresponds to a 49% recovery of this isomer. The (−)enantiomer of the acid was isolated in the same manner as describedabove for the (+) acid. The resolved salt was suspended in ether andwater, the pH lowered with concentrated H₂SO₄, and the product extractedin the organic phase.

Example 5 A. Preparation of(−)4-Chlorophenyl-(3-trifluoromethylphenoxy)-acetyl Chloride

A 2-L evaporation flask with magnetic stirrer, Claissen adapter, potthermometer and a reflux condenser routed to a gas scrubber was chargedwith (−)4-chlorophenyl-(3-trifluoromethylphenoxy)-acetic acid (143 g,0.42 mole based on 97% purity) and CHCl₃ (170 ml) and heated to boilingin order to dissolve. SOCl₂ (38 ml, 62.1 gm, 0.52 mole) was added. Themixture was heated to reflux (68° C. final) for 4.5 hours and thenstripped of volatiles to obtain 151 g yellow, turbid liquid (103%apparent yield). The material was used in the next step without furtherpurification.

B. Preparation of (+)4-Chlorophenyl-(3-trifluoromethylphenoxy)-acetylChloride

A 3-L evaporation flask with magnetic stirrer, Claissen adapter, potthermometer and a reflux condenser routed to a gas scrubber was chargedwith (+)4-chlorophenyl-(3-trifluoromethylphenoxy)-acetic acid (131 g,0.37 mole) and CHCl₃ (152 ml) and heated to boiling in order todissolve. SOCl₂ (35 ml, 56.5 g, 0.48 mole) was added. The mixture washeated to reflux (70° C. final) for 4 hours and then stripped ofvolatiles to obtain 139 g liquid. The material was used in the next stepwithout further purification.

Example 6 A. Preparation of (−)2-Acetamidoethyl4-Chlorophenyl-(3-trifluoromethylphenoxy)-acetate

A 3-L round-bottom flask with magnetic stirrer, pot thermometer, under anitrogen atmosphere and in an ice-water bath was charged with DMF (420ml), pyridine (37 ml, 36 g, 0.46 mole) and N-acetoethanolamine (39 ml,43 g, 0.42 mole). The mixture was cooled to 0° to 5° C. and a solutionof crude (−)4-chlorophenyl-(3-trifluoromethylphenoxy)-acetyl chloride(151 gm, 0.42 mole based on 100% yield of previous step) in ether (170ml) was added over a 40 min. period so as to maintain the pottemperature below 13° C. The mixture was stirred at ambient temperaturefor 16 hours and dissolved by adding water (960 ml) followed by ethylacetate (630 ml). The water addition proceeded exothermically raisingthe temperature from 24° to 34° C. Ethyl acetate addition caused atemperature drop to 30° C. The layers were separated and the aqueousphase extracted once with ethyl acetate (125 ml). The combined organiclayers were extracted once with 7% (w:w) aqueous NaHCO₃ (125 ml) andfive times with 60 ml portions of water and then twice with 60 mlportions of 25% (w:w) aqueous NaCl. The product was dried over MgSO₄ (42g) and filtered through Whatman #1 filter paper. Solvent was strippedusing a rotary evaporator to obtain 160 g of a yellow syrupcorresponding to an 80% yield based on the proton NMR that shows 87%product, 8% EtOAc, 4% non-brominated amide, and 1% DMF. This syrup wasdissolved in MTBE (225 ml) at ambient temperature and chilled (−15° C.)85% hexanes (400 ml) was added with stirring. Two liquids formed, thencrystals, then the mixture formed a solid. The solid mass was scrapedonto a Buchner funnel fitted with Whatman #1, packed down and washedthree times with 100 ml portions of 1:1 (v:v) MTBE:hexanes to obtain 312g wet product which dries to 127 gm, corresponding to a 73% yield.

B. Preparation of (+)2-Acetamidoethyl4-Chlorophenyl-(3-trifluoromethylphenoxy)-acetate

A 3-L round-bottom flask with magnetic stirrer, pot thermometer, under anitrogen atmosphere and in an ice-water bath was charged with DMF (365ml), pyridine (33 ml, 32.3 g, 0.41 mole) and N-acetoethanolamine (34 ml,38.1 g, 0.37 mole). The mixture was cooled to 0° to 5° C. and a solutionof crude (+)4-chlorophenyl-(3-trifluoromethylphenoxy)-acetyl chloride(139 gm, 0.37 mole based on 100% yield of previous step) in ether (155ml) was added over a 25 min. period so as to maintain the pottemperature below 13° C. The mixture was stirred at ambient temperature40 hours and dissolved by adding water (850 ml) followed by ethylacetate (550 ml). The water addition proceeded exothermically raisingthe temperature from 24° to 34° C. Ethyl acetate addition caused atemperature drop to 30° C. The layers were separated and the aqueousphase extracted once with ethyl acetate (110 ml). The combined organiclayers were washed twice with 55 ml portions of water and then fivetimes with 55 ml portions of 25% (w:w) aqueous NaCl and dried over 30 gMgSO₄ and filtered through Whatman #1 filter paper. Solvent was strippedusing a rotary evaporator to obtain 168 g yellow liquid corresponding toan 86% yield based on the proton NMR that shows 79% product, 9% EtOAc,8% non-brominated amide, and 4% DMF. The product was crystallized in an800-ml beaker by dissolving in MTBE (200 ml) at ambient temperature,cooling at −15° for 1.4 hours, adding 200 ml 85% hexanes and thenchilling 1 hour. The solid mass was scraped out onto a Buchner funnelfitted with Whatman #1, packed down and washed once with 1:1 (v:v)MTBE:hexanes (100 ml) to obtain 201 gm wet product. The product wasdried under nitrogen flow and triturated with 85% hexanes (700 ml) usingan overhead stirrer. The material was filtered and dried to obtain 87 gmproduct. [α]_(D)+2.769° (c.=0.048 in methyl alcohol). [α]_(D)−2.716°(c.=0.049 in methyl alcohol). The (+) and (−) enantiomers were alsoanalyzed by HPLC using a 250×4.6 mm R,R-WhelkO-1 column at ambienttemperature. Injected samples were 20 μl of 2 mg/ml solutions of thesamples in ethanol. The column was eluted with 60:40, isopropanol:hexaneat a flow of 1 ml/min. Detection was at 220 nm. The (+) enantiomereluted at 5.0 to 5.2 min. and the (−) enantiomer at 5.7 to 5.9 min.

Example 7

This example relates to the inhibition of cytochrome P450 2C9 (CYP2C9)by the compounds of the present invention.

Tolbutamide hydroxylation activity (100 μM ¹⁴C-tolbutamide; 1 mM NADPH)was assayed in pooled human liver microsomes (0.6 mg protein/ml)) for 60minutes at 37° C. both with and without test compounds. Racemichalofenic acid, (−) halofenic acid and (+) halofenic acid were tested(0.25 μM to 40 μM). As shown in FIG. 1, racemic halofenic acid inhibitedCYP2C9-mediated tolbutamide hydroxylation activity in human livermicrosomes with an apparent IC₅₀ of 0.45 μM. A substantial differencewas noted in the ability of the enantiomers of halofenic acid to inhibitCYP2C9. The (+) halofenic acid had an apparent IC₅₀ of 0.22 μM whereasthe (−) halofenic acid was almost 20-fold less potent with an apparentIC₅₀ of 3.6 μM.

Example 8

This example relates to the time course of glucose-lowering for thecompounds of the present invention.

A. Material and Methods

Male, 9-10 weeks old, C57BL/63 ob/ob mice were purchased from TheJackson Laboratory (Bar Harbor, Me., USA). Animals were housed (4-5mice/cage) under standard laboratory conditions at 22° C. and 50%relative humidity, and were maintained on a diet of Purina rodent chowand water ad libitum. Prior to treatment, blood was collected from thetail vein of each animal. Mice that had non-fasting plasma glucoselevels between 300 and 500 mg/dl were used. Each treatment groupconsisted of 10 mice that were distributed so that the mean glucoselevels were equivalent in each group at the start of the study. Micewere dosed orally once by gavage with either vehicle, racemic halofenate(250 mg/kg), (−) halofenate (250 mg/kg) or (+) halofenate (250 mg/kg).All compounds were delivered in a liquid formulation contained 5% (v/v)dimethyl sulfoxide (DMSO), 1% (v/v) tween 80 and 2.7% (w/v)methylcellulose. The gavage volume was 10 ml/kg. Blood samples weretaken at 1.5, 3, 4.5, 6, 7.5, 9 and 24 hour after the dose and analyzedfor plasma glucose. Plasma glucose concentrations were determinedcolorimetrically using glucose oxidase method (Sigma Chemical Co, St.Louis, Mo., USA). Significance difference between groups (comparingdrug-treated to vehicle-treated or between drug-treated groups) wasevaluated using Student unpaired t-test.

B. Results

As illustrated in FIG. 2, racemic halofenate significantly reducedplasma glucose concentrations at most of the timepoints with the peakactivity at 9 hours. (−) halofenate showed a plasma glucose reduction asearly as 1.5 hours and reached its peak activity at 3 hours. The plasmaglucose concentrations remained low up to 24 hours. (+) halofenate didnot show significant activity until 4.5 hours and the peak activity wasat 7.5 hours. Plasma glucose started to rebound afterward. There weresignificant differences between (−) and (+) enantiomers of halofenate atthe 3 and 24-hour timepoints. The activity of the (−) halofenate wasmore rapid onset and sustained longer.

Example 9

This example relates to the Glucose lowering activity of the compoundsof the present invention.

A. Materials and Methods

Male, 8-9 weeks old, C57BL/6J ob/ob mice were purchased from The JacksonLaboratory (Bar Harbor, Me., USA). Animals were housed (4-5 mice/cage)under standard laboratory conditions at 22° C. and 50% relativehumidity, and were maintained on a diet of Purina rodent chow and waterad libitum. Prior to treatment, blood was collected from the tail veinof each animal. Mice that had non-fasting plasma glucose levels between300 and 520 mg/dL were used. Each treatment group consisted of 10 micethat were distributed so that the mean glucose levels were equivalent ineach group as the start of the study. Mice were dosed orally by gavageonce a day for 5 days with either vehicle, racemic halofenate (250mg/kg), (−) halofenate (125 and 250 mg/kg) or (+) halofenate (125 and250 mg/kg). Racemic halofenate was delivered in 2.7% (w/v)methylcellulose and both the (−) enantiomer and (+) enantiomer weredelivered in a liquid formulation contained 5% (v/v) dimethyl sulfoxide(DMSO), 1% (v/v) tween 80 and 2.7% (w/v) methylcellulose. The gavagevolume was 10 ml/kg. Blood samples were taken at 3, 6, 27, 30 and 120hour after the first dose and analyzed for plasma glucose and insulin.The animals were fasted overnight (14 hours) before the 120 hourssampling. Plasma glucose concentrations were determined colorimetricallyusing glucose oxidase method (Sigma Chemical Co, St. Louis, Mo., USA).Plasma insulin concentrations were determined by using the Rat InsulinRIA Kit from Linco Research Inc. (St. Charles, Mo., USA). Significancedifference between groups (comparing drug-treated to vehicle-treated)was evaluated using Student unpaired t-test.

B. Results

As illustrated in FIG. 3, (−) halofenate significantly reduced plasmaglucose concentrations at 6, 27 and 30 hours. (−) halofenate at bothdosage levels significantly lowered plasma glucose concentrations at 6,27 and 30 hours. The high-dose (250 mg/kg) was also active at 3 hours.(+) halofenate at 125 mg/kg showed plasma glucose reduction at 6 and 27hours, where at 250 mg/kg, lowered plasma glucose concentrations wereobserved at 3, 6, 27 and 30 hours. Plasma insulin levels are shown inFIG. 4. Racemic halofenate significantly reduced insulin at 6 and 27hours. Plasma insulins were significantly reduced in the (−) halofenategroup at 27 hours at both doses and was significantly reduced at 30hours in the animals treated with 250 mg/kg/day. (+) halofenatesignificantly reduced insulin at 27 and 30 hours at both doses. At 125mg/kg/day a significant reduction was also observed after 6 hours. Afterfasting overnight (at 120 hours), all treatments reduced plasma glucoseconcentrations significantly (FIG. 5). Plasma insulins weresignificantly reduced in all halofenate treated groups except the (+)halofenate at 125 mg/kg/day (FIG. 6).

Example 10

This example relates to the improvement in Insulin Resistance andImpaired Glucose Tolerance for the compounds of the present invention.

Materials and Methods

Male, 8-9 weeks old Zucker fa/fa rats (Charles River,) were housed (2-3rats/cage) under standard laboratory conditions at 22° C. and 50%relative humidity, and were maintained on a diet of Purina rodent chowand water ad libitum. Prior to treatment, rats were assigned to 6 groupsbased on body weight. Each treatment group consisted of 8 rats. Ratswere dosed orally once by gavage with either vehicle, racemic halofenate(100 mg/kg), (−) halofenate (50 or 100 mg/kg) or (+) halofenate (50 or100 mg/kg). All compounds were delivered in a liquid formulationcontained 5% (v/v) dimethyl sulfoxide (DMSO), 1% (v/v) tween 80 and 2.7%(w/v) methylcellulose. The gavage volume was 10 ml/kg. All rats receivedan oral glucose challenge (1.9 g/kg) 5.5 hours after the treatment and 4hours after withdrawal of the food. Blood samples were taken at 0, 15,30, 60, 90, 120, and 180 minutes following the glucose challenge forplasma glucose measurement. The vehicle, (−) halofenate (50 mg/kg) and(+) halofenate (50 mg/kg) groups were subjected to an insulin challengefollowing daily gavage of the respective treatments for 5 days. On day5, rats received the intravenous insulin (0.75 U/kg) 5.5 hours after thelast dose and 4 hours after withdrawal of the food. Blood samples weretaken at 3, 6, 9, 12, 15 and 18 minutes following the insulin injectionfor plasma glucose measurement. Plasma glucose concentrations weredetermined colorimetrically using glucose oxidase method (Sigma ChemicalCo, St. Louis, Mo., U.S.A.). Significance difference between groups(comparing drug-treated to vehicle-treated or between drug-treatedgroups) was evaluated using Student unpaired t-test.

B. Results

As illustrated in FIG. 7A, Zucker fatty rats with Impaired GlucoseTolerance had lower plasma glucose levels after a glucose challengefollowing treatment with halofenate. The (−) halofenate was the mosteffective in lowering the glucose and had an effect that persistedlonger than the racemate or (+) enantiomer. FIG. 7B shows theincremental area under the curve (AUC) for all the treatment groups. Theanimals treated with the (−) halofenate showed significant reductions inthe glucose area relative to vehicle-treated controls. Although the AUCwas decreased in the groups treated with the racemate or (+) halofenate,the effects were not as great as in the (−) halofenate-treated rats andthe differences were not statistically significant.

Changes in insulin sensitivity were assessed by monitoring the fall inglucose after an intravenous injection of insulin. The slope of the lineis a direct indication of the insulin sensitivity of the test animal. Asshown in FIG. 8, the insulin sensitivity was improved significantlyafter 5 days of treatment with (−) halofenate compared to thevehicle-treated controls (p<0.01) and animals treated with (+)halofenate (p<0.05). Treatment with (+) halofenate had a small effect oninsulin sensitivity that was not significantly different from thevehicle-treated control (p=0.083). Treatment with (−) halofenatesubstantially reduced the insulin resistance in the Zucker fatty rat, awell-established model of Impaired Glucose Tolerance and insulinresistance.

Example 11

This example relates to the lipid lowering activity of the compounds ofthe present invention.

A. Materials and Methods

Male Zucker diabetic fatty (ZDF) rats were obtained from GMILaboratories (Indianapolis, Ind.) at 9 weeks of age. Vehicle orenantiomers of halofenate administered by oral gavage on a daily basisstarting at 74 days of age. Initial blood samples were obtained foranalysis one day before treatment and at the indicated times in thetreatment protocol. Blood was analyzed for plasma triglyceride andcholesterol by standard techniques.

B. Results

In experiment I animals received a dose of 25 mg/kg/day. As shown inFIG. 9A and FIG. 9B, a significant decrease in plasma cholesterol wasnoted only in animals treated with the (−) halofenate after 7 and 13days of treatment. In Experiment II, animals at 107 days of age receiveddaily doses of either 12.5 mg/kg/day or 37.5 mg/kg/day of the (−) and(+) enantiomers of halofenate. As shown in FIG. 10A and FIG. 10B, theplasma cholesterol was significantly lower on the high dose after 7 daysbut not after 14 days of treatment with the (+) halofenate. In contrast,for the (−) halofenate at the low dose, a significant decrease incholesterol was observed after 7 days. At the high dose a much greaterdecline in plasma cholesterol was noted that was apparent both after 7and 14 days of treatment. As shown in FIG. 11A and FIG. 11B, asignificant decrease in plasma triglyceride was also noted 7 days aftertreatment at the high dose which was of greater magnitude in animalstreated with the (−) enantiomer of halofenate.

Example 12

This example relates to the glucose lowering activity of (±) halofenateanalogs and (−) halofenate analogs.

A. Materials and Methods

Male, 8-9 weeks old, C57BL/6J ob/ob mice were purchased from The JacksonLaboratory (Bar Harbor, Me., USA). Animals were housed (4-5 mice/cage)under standard laboratory conditions at 22±3° C. temperature and 50±20%relative humidity, and were maintained on a diet of Purina rodent chowand water ad libitum. Prior to treatment, blood was collected from thetail vein of each animal. Mice that had non-fasting plasma glucoselevels between 250 and 500 mg/dl were used. Each treatment groupconsisted of 8-10 mice that were distributed so that the mean glucoselevels were equivalent in each group at the start of the study. Micewere dosed orally by gavage once a day for 1-3 days with either vehicle,(−) halofenic acid, (±) analog 14, 29, 33, 34, 35, 36, 37, or 38 at 125mg/kg or (−) analog 29, 36, 37 or 38 at 150 mg/kg. Compounds weredelivered in a liquid formulation containing 5% (v/v) dimethyl sulfoxide(DMSO), 1% (v/v) tween 80 and 0.9% (w/v) methylcellulose. The gavagevolume was 10 ml/kg. Blood samples were taken at 6 hours after the eachdose and analyzed for plasma glucose. Food intake and body weight weremeasured daily. Plasma glucose concentrations were determinedcolorimetrically using glucose oxidase method (Sigma Chemical Co, St.Louis, Mo., USA). Significant difference between groups (comparingdrug-treated to vehicle-treated) was evaluated using the Studentunpaired t-test.

B. Results

As illustrated in Table 2, compounds were evaluated in 5 differentexperiments. Single dose (−) halofenic acid significantly reduced plasmaglucose concentrations at 6 hours. Analog 14 significantly loweredplasma glucose concentrations at 6, 30 and 54 hours. Analog 33significantly lowered plasma glucose concentrations at 6 and 54 hours.Analog 29 and 38 significantly lowered plasma glucose concentrations at6, 30 and 54 hours. Analog 35 and 36 significantly lowered plasmaglucose concentrations at 30 and 54 hours. Analog 37 significantlylowered plasma glucose concentrations at 54 hours. Single dose (−)analogs 29, 36, 37 and 38 significantly reduced plasma glucoseconcentrations at 6 hours. Compound treatments did not affect theanimal's food intake and body weight.

TABLE 1 (±) and (−) Halofenate Analogs. Compounds Described in Referenceto Formula II. Cmpd No. X CX₃ R² halofenic Cl CF₃ H acid 14 F CF₃(CH₂)₂NHAc 29 Br CF₃ (CH₂)₂NHAc 33 Cl CF₃ (CH₂)₃CH₃ 35 Cl CF₃(CH₂)₂N(CH₃)₂ 36 Cl CF₃ (CH₂)₂NHCOPh 37 Cl CF₃ CH₂CONH₂ 38 Cl CF₃CH₂CON(CH₃)₂

TABLE 2 Glucose-Lowering Activities of (±)Halofenate and (−)HalofenateAnalogs. 6 hours 30 hours 54 hours Predose P P P Glucose Glucose VALUEGlucose VALUE Glucose VALUE (mg/dl) (mg/dl) vs. veh (mg/dl) vs. veh(mg/dl) vs. veh Vehicle 313 ± 18   303 ± 19.8 NA NA (−)halofenic 312.9 ±17.7 163.8 ± 11.8 0.0011 NA NA acid Vehicle 360.2 ± 27.8 405.8 ± 25.8356.0 ± 27.6 386.1 ± 20.6 (±)Analog 14 361.0 ± 17.1 328.9 ± 34.1 0.0444267.0 ± 21.3 0.0099 293.0 ± 29.4 0.0092 Vehicle 291.6 ± 18.5 363.0 ±25.1 340.8 ± 30.0 351.5 ± 23.8 (±)Analog 33 292.0 ± 19.1 227.5 ± 13.20.0001 298.0 ± 15.3 0.1119 286.6 ± 9.9  0.0125 Vehicle 387.1 ± 14.3371.5 ± 24.2 326.2 ± 22.5 374.0 ± 37.9 (±)Analog 29 387.1 ± 16.0 299.7 ±24.5 0.0259 237.4 ± 14.9 0.0020 293.3 ± 9.7  0.0268 (±)Analog 35 387.0 ±18.0 319.6 ± 26.7 0.0834 276.8 ± 17.6 0.0504 286.2 ± 31.5 0.0458(±)Analog 37 387.4 ± 18.8 345.4 ± 19.7 NS 312.5 ± 21.7 NS 285.1 ± 14.70.0210 Vehicle 329.6 ± 16.1 361.8 ± 23.2 346.5 ± 24.6 379.2 ± 24.4(±)Analog 36 329.7 ± 17.6 300.5 ± 27.3 0.0522 249.7 ± 8.6  0.0008 272.2± 18.4 0.0013 (±)Analog 38 329.4 ± 18.9 303.2 ± 18.2 0.0312 245.6 ± 15.60.0014 243.1 ± 10.6 0.0000 Vehicle 373.0 ± 13.6 405.8 ± 33.7 NA NA(−)Analog 36 373.2 ± 15.5 281.1 ± 18.2 0.0019 NA NA (−)Analog 37 373.4 ±16.1 271.7 ± 22.5 0.0018 NA NA (−)Analog 38 373.4 ± 16.1 251.2 ± 23.60.0007 NA NA (−)Analog 29 372.2 ± 17.1 333.5 ± 16.1 0.0353 NA NA

Example 13

This example relates to a comparison between the activities of (−)halofenate and (+) halofenate.

A. Materials and Methods

Male 8-9 week old ZDF rats were purchased from Genetic Models, Inc.(Indianapolis, Ind.). Animals were housed (3 rats/cage) under standardlaboratory conditions at 22±3° C. temperature and 50±20% relativehumidity, and were maintained on a diet of Purina rodent chow and waterad libitum. Prior to treatment, blood was collected from the tail veinof each animal. Rats that had 4-hour fasting plasma glucose levelsbetween 200 and 500 mg/dL were used. Each treatment group consisted of8-10 rats that were distributed so that the mean glucose levels wereequivalent in each group at the start of the study. Rats were dosedorally by gavage once a day for 3 days with either vehicle, (−)halofenate or (+) halofenate at 50 mg/kg. Compounds were delivered in aliquid formulation containing 5% (v/v) dimethyl sulfoxide (DMSO), 1%(v/v) tween 80 and 0.9% (w/v) methylcellulose. The gavage volume was 5ml/kg. Blood samples were taken at 5 hours post dose on day 2 and 3.Plasma glucose concentrations were determined colorimetrically usingglucose oxidase method (Sigma Chemical Co, St. Louis, Mo., USA).Significant difference between groups (comparing drug-treated tovehicle-treated) was evaluated using the Student unpaired t-test.

B. Results

Oral administration of (−) halofenate at 50 mg/kg significantly reducedplasma glucose concentrations, while (+) halofenate at the same dosagelevels failed to reduce plasma glucose concentrations as compared tovehicle-treated animals (FIG. 12).

Example 14

This example relates to a pharmacokinetic study of (±) halofenate and(−) halofenate.

A. Materials and Methods

Male 225-250 g SD rats were purchased from Charles River. Animals were °housed (3 rats/cage) under standard laboratory conditions at 22±3° C.temperature and 50±20% relative humidity, and were maintained on a dietof Purina rodent chow and water ad libitum. A catheter was placed in theleft carotid artery under sodium pentobarbital (50 mg/kg i.p.) andanimals were allowed to recover for 2 days before treatment. Single doseof (±) halofenate or (−) halofenate at 50 mg/kg were administered byoral gavage. Compounds were delivered in a liquid formulation containing5% (v/v) dimethyl sulfoxide (DMSO), 1% (v/v) tween 80 and 0.9% (w/v)methylcellulose. The gavage volume was 5 ml/kg. Blood samples werecollected at 1, 2, 4, 6, 8, 12, 24, 48, 72, 96 and 120 hours post dose.The plasma samples were analyzed for each enantiomeric acid ((−)halofenic acid and (+) halofenic acid) by a chiral specific HPLC assay,since the esters are prodrugs, which are designed to convert to theirrespective enantiomeric acids in vivo.

B. Results

After oral administration of (±) halofenate, both (−) halofenic acid and(+) halofenic acid were detected in the plasma samples. As shown inTable 3, it appeared that the two enantiomeric acids had differentdispositional profiles. The elimination of (−) halofenic acid was muchslower than (+) halofenic acid. As a result, the AUC of (−) halofenicacid was significantly higher than the AUC for (+) halofenic acid,4708.0 vs. 758.0 μg·h/mL and the terminal half-life was 46.8 vs. 14.3hours.

After oral administration of (−) halofenate, the dispositional profileof (−) halofenic acid was basically identical to the administration of(±) halofenate as the terminal half-life is the same (Table 2). The Cmaxand AUC of (−) halofenic acid were proportionally higher simply due tohigher amount of (−) halofenate administered (Table 3). (+) halofenicacid was also detected in the plasma but the concentration was muchlower than (−) halofenic acid. It is speculated that (+) halofenic acidwas formed in vivo since the terminal half-life (T_(1/2)) of both acidswas similar.

These results suggest the use of (−) halofenate is more desirable sincethe AUC of (−) halofenic acid was significantly higher than the AUC for(+) halofenic acid.

TABLE 3 Pharmacokinetic Analysis of (−) Halofenate (−Enantiomer) and (+)Halofenate (+Enantiomer). Drug administered (−) Halofenate (n = 3) (±)Halofenate (n = 1) Enantiomer − + − + Dose administered* 50 mg/kg 0(metabolite) 25 mg/kg 25 mg/kg C_(max) (μg/mL) 114.6 ± 29.7 2.4 ± 0.565.2 30.5 T_(max) (hours)  8-12  6-12 12 6 AUC  7159 ± 1103 164.3 ±79.3  4708 758 (μg · h/mL) T_(1/2) (hours) 46.4 ± 4.7 41.7 ± 11.8 46.814.3 The dose of each enantiomer in (±) halofenate is 50% of the totaldose of the racemic mixture.

TABLE 4 Plasma Concentrations of (−) Halofenic Acid and (+) HalofenicAcid Following a Single Dose of (−) Halofenate. Compound Analyzed(μg/mL) Time (−) halofenic acid (+) halofenic acid (hour) Rat 8 Rat 9Rat 11 Rat 8 Rat 9 Rat 11 0 BQL BQL BQL BQL BQL BQL 1 81.2 23.7 61.01.12 BQL BQL 2 100.1 30.4 87.8 1.27 BQL 1.09 4 122.3 36.9 94.5 1.67 BQL1.95 6 128.3 56.5 116.3 2.96 BQL 1.73 8 128.2 79.0 127.8 2.58 BQL 2.0612 135.3 80.6 104.8 2.85 2.23 2.08 24 82.5 73.1 66.5 2.22 1.29 1.86 4856.2 44.5 47.1 1.64 1.03 1.14 72 39.7 37.4 30.8 1.25 BQL BQL 96 31.1 N/A24.6 BQL N/A BQL 120 20.3 N/A N/A BQL N/A N/A *BQL = Below QuantifiableLimit <1.00 μg/mL N/A = Sample not available

Example 15

This example relates to the prevention of the development of diabetesand the alleviation of hypertriglyceridemia by (−) halofenate.

A. Materials and Methods

Male, 4 weeks old, C57BL/6J db/db mice were purchased from The JacksonLaboratory (Bar Harbor, Me., USA). Animals were housed (5 mice/cage)under standard laboratory conditions at 22±3° C. temperature and 50±20%relative humidity, and were maintained on a powder diet of Purina rodentchow (#8640) and water ad libitum. Prior to treatment, blood wascollected from the tail vein of each animal for plasma glucose, insulinand triglyceride concentrations. Mice were distributed so that the meanglucose levels and body weight were equivalent in each group at thestart of the study. The control group (20 mice) was put on powder chowmixed with 5% sucrose and the treatment group (20 mice) was put onpowder chow mixed with 5% sucrose and (−) halofenate. The amount of (−)halofenate in the chow was adjusted continuously according the animal'sbody weight and food intake to meet the target dosage of 150 mg/kg/day.Blood samples were taken at 8-10 AM once a week for 9 weeks undernon-fasting condition. Food intake and body weight were measured every1-3 days. Plasma glucose and triglyceride concentrations were determinedcolorimetrically using kits from Sigma Chemical Co (No. 315 and No. 339,St. Louis, Mo., USA). Plasma insulin levels were measured using RIAassay kit purchased from Linco Research (St. Charles, Mo.). Significantdifferences between groups (comparing drug-treated to vehicle-treated)was evaluated using Student unpaired t-test.

B. Results

C57BL/6J db/db mice at 4 weeks of age are in a pre-diabetic state. Theirplasma glucose concentrations are normal, but the plasma insulinconcentrations are significantly elevated. As illustrated in FIG. 13,the plasma glucose concentrations in both groups were normal at thestart of the experiment. Following the natural course of diabetesdevelopment, plasma glucose levels in the control group increasedprogressively as the animals aged, while the increase of plasma glucoselevels in the (−) halofenate treated group was prevented orsignificantly delayed. As depicted in FIG. 15, about 30% of mice did notdevelop diabetes in the (−) halofenate treated group when diabetes isdefined as plasma glucose levels>250 mg/dl. On the other hand, none ofthe mice in the control group was free of diabetes by the age of 10weeks. Consistent with the plasma glucose finding, plasma insulin in thecontrol group decreased progressively, indicating deterioration of theability of the pancreas to secret insulin. (−) halofenate treatmentmaintained the plasma insulin concentration, indicating prevention ofthe deterioration of pancreatic function (FIG. 14).

FIG. 16 shows progression of the plasma triglyceride concentrationsversus age in C57BL/6J db/db mice. (−) halofenate administrationalleviated the increase of plasma triglyceride concentration over thecourse of the experiment.

Example 16

This example describes the preparation of (−)2-Acetamidoethyl4-Chlorophenyl-(3-trifluoro methylphenoxy)-acetate ((−) halofenate).

4-Chlorophenylacetic acid was combined with 1,2-dichloroethane and theresulting solution was heated to 45° C. Thionyl chloride was added tothe reaction mixture, which was heated at 60° C. for 18 hours. Thereaction was allowed to cool to room temperature and was then addedslowly to a solution of N-acetylethanolamine in dichloromethane. Afterstirring 30 min., the reaction was quenched with aqueous potassiumcarbonate and sodium thiosulfate. The organic layer was washed withwater, dried over magnesium sulfate and filtered. Removal of the solventby rotary evaporation provided N-acetylaminoethyl2-bromo-2-(4-chlorophenyl)acetate as an oil.

3-Hydroxybenzotrifluoride was added to a solution of potassium hydroxidein isopropanol. N-acetylaminoethyl 2-bromo-2-(4-chlorophenyl)acetate inisopropanol was added to the isopropanol/phenoxide solution and stirredat room temperature for 4 hours. The isopropanol was removed by vacuumdistillation, and the resulting slush was dissolved in ethyl acetate andwashed twice with water and once with brine. After drying over magnesiumsulfate and filtration, the solvent was removed to give crude product asan oil. The crude product was dissolved in hot toluene/hexanes (1:1 v/v)and cooled to between 0 and 10° C. to crystallize the product. Thefilter cake was washed with hexanes/toluene (1:1 v/v) and then driedunder vacuum at 50° C. The isolated solid was dissolved in hot 1:6 (v/v)isopropanol in hexanes. After cooling, the pure racemic 2-Acetamidoethyl4-Chlorophenyl-(3-trifluoro methylphenoxy)-acetate formed as acrystalline solid. The solid was collected by filtration, the filtercake washed with 1:6 (v/v) isopropanol in hexanes and dried under vacuumat 50° C.

The racemic compound was dissolved in a solution of 20% isopropanol(IPA) and 80% hexane at 2.5% (wt/wt). The resulting solution was passedover a Whelk-O R,R Chiral Stationary Phase (CSP) in continuous fashionuntil >98% ee extract could be removed. The solvent was evaporated fromthe extract under reduced pressure to provide (−)2-Acetamidoethyl4-Chlorophenyl-(3-trifluoro methylphenoxy)-acetate. (The SimulatedMoving Bed resolution was conducted by Universal Pharm Technologies LLCof 70 Flagship Drive, North Andover, Mass. 01845.)

Example 17

This example relates to the lowering of plasma uric acid levels throughthe administration of (−) halofenate.

A. Materials and Methods

Male SD rats, weight 275-300 g were purchased from Charles River.Animals were housed (3 rats/cage) under standard laboratory conditionsat 22±3° C. temperature and 50±20% relative humidity, and weremaintained on a powder diet of Purina rodent chow (#8640) and water adlibitum. To establish a hyperuricemic state, animals were put on a dietcontaining 2.5% (w/w) of oxonic acid (Sigma Chemical Co, St. Louis, Mo.,USA) throughout the experiment. Oxonic acid elevates plasma uric acid byinhibiting uricase. Rats were screened for plasma uric acid levels 3days after they were placed on the diet, and those that had extremeplasma uric acid levels were excluded. Rats were assigned to one ofthree groups and the mean uric acid levels were equivalent in eachgroup. Rats were dosed orally by gavage once a day for 3 days witheither vehicle, (−) halofenate or (+) halofenate at 50 mg/kg. On the4^(th) day, respective rats received (−) halofenate or (+) halofenate at100 mg/kg and all rats received an i.p. injection of oxonic acid (250mg/kg) 4 hours after the oral gavage. (−) halofenate and (+) halofenatewere delivered in a liquid formulation containing 5% (v/v) dimethylsulfoxide (DMSO), 1% (v/v) tween 80 and 0.9% (w/v) methylcellulose.Oxonic acid was delivered in a liquid formulation containing 0.9% (w/v)methylcellulose. The gavage and injection volumes were 5 ml/kg. Bloodsamples were taken at 6 hours post oral gavage on day 4. Plasma uricacid levels were determined colorimetrically using the Infinity UricAcid Reagent (Sigma Chemical Co, St. Louis, Mo., USA). Significantdifference between the groups (comparing drug-treated tovehicle-treated) was evaluated using the Student unpaired t-test.

B. Results

As shown in FIG. 17, oral administration of (−) halofenate significantlyreduced plasma uric acid levels. (+) halofenate also lowered plasma uricacid levels, but it was not statistically significant.

Example 18

This example relates to the inhibition of cytochrome P450 isoforms bythe compounds of the present invention.

A. Materials and Methods

The following probe substrates were used to investigate the inhibitorypotential of the test article on the cytochrome P450 isoforms 1A2, 2A6,2C9, 2C19, 2D6, 2E1 and 3A4: 100 μM phenacetin (CYP1A2), 1 μM coumarin(CY)2A6), 150 μM tolbutamide (CYP2C9), 50 μM S-mephenyloin (CYP2C19), 16μM dextromethorphan (CYP2D6), 50 μM chlorzoxazone (CYP2E1), and 80 μMtestosterone (CYP3A4). The activity of each isoform was determined inhuman hepatic microsomes in the presence and absence of the testarticle.

Unless otherwise noted, all incubations were conducted at 37° C. Thesample size was N=3 for all test and positive control conditions and N=6for all vehicle control conditions. (−) Halofenic acid (MW=330) wasprepared at room temperature as 1000× stocks in methanol, then dilutedwith Tris buffer to achieve final concentrations of 0.33, 1.0, 3.3, 10and 33.3 μM, each containing 0.1% methanol. A vehicle control (VC)consisting of microsomes and substrate in Tris buffer containing 0.1%methanol without the test article was included for all experimentalgroups. Positive control (PC) mixtures were prepared using the followingknown CYP450 inhibitors: 5 μM furafylline (CYP1A2), 250 μMtranylcypromine (CYP2A6), 50 μM sulfaphenazole (CYP2C9), 10 μMomeprazole (CYP2C19), 1 μM quinidine (CYP2D6), 100 μM 4-methylpyrazole(CYP2E1), and 5 μM ketoconazole (CYP3A4). A chromatographic interferencecontrol (CIC) was included to investigate the possibility ofchromatographic interference by the test article and its metabolites.The test article (at 33.3 μg/mL) was incubated with 1× microsomalprotein, 1×NRS, and 10 μL of an appropriate organic for an appropriatetime period as described below.

Stable, frozen lots of pooled adult male and female hepatic microsomesprepared by differential centrifugation of liver homogenates were usedin this study (see, e.g., Guengerich, F. P. (1989). Analysis andcharacterization of enzymes. In Principles and Methods of Toxicology (A.W. Hayes, Ed.), 777-813. Raven Press, New York.). Incubation mixtureswere prepared in Tris buffer to contain microsomal protein (1 mg/mL),each concentration of the probe substrates (as 100× stocks), and thetest article (at each concentration) or PC as appropriate for eachisoform. After a 5-minute preincubation at 37° C., NADPH regeneratingsystem (NRS) was added to initiate the reactions, and the samples wereincubated at 37° C. for the following time periods: 30 minutes forphenacetin (CYP1A6), 20 minutes for coumarin (CYP2A6), 40 minutes fortolbutamide (CYP2C9), 30 minutes for S-mephenyloin (CYP2C19), 15 minutesfor dextromethorphan (CYP2D6), 20 minutes for chlorozoxazone (CYP2E1),and 10 minutes for testosterone (CYP3A4). Incubation reactions wereterminated at the appropriate time with the addition of an equal volumeof methanol, except for the incubations with S-mephenyloin, which wereterminated with the addition of 100 μL of perchloric acid. Allsubstrates were evaluated near their respective K_(m) concentrations, aspreviously indicated.

After each incubation, the activities of the P450 isoforms weredetermined by measuring the rates of metabolism for the respective probesubstrates. The metabolites monitored for each probe substrate were asfollows: acetaminophen for CYP1A2; 7-hydroxycoumarin for CYP2A6;4-hydroxytolbutramide for CYP2C9; 4-hydroxymephenyloin for CYP2C19;dextrorphan for CYP2D6; 6-hydroxychlorzoxazone for CYP2E1; and6β-hydroxytestosterone for CYP3A4. Activities were analyzed using HPLC(In Vitro Technologies, Inc., Baltimore, Md.).

Inhibition was calculated using the following equation:Percent Inhibition=[(vehicle control−treatment)/vehicle control]×100

Percent inhibition data for the test article was presented in a tabularformat. Descriptive statistics (mean and standard deviation) of eachtest article concentration were calculated, then presented to showinhibitory potency. IC₅₀ values were also calculated for the testarticle using a 4-parameter curve fitting equation in Softmax 2.6.1.

Measures of time, temperature, and concentration in this example areapproximate.

B. Results

The results for each of the 7 isoforms of cytochrome P450, expressed asmetabolic activity and percentage of inhibition, are presented in Tables5-8. (−) Halofenic acid inhibited 4-hydroxytolbutamide production(CYP2C9, IC50=11 μM) and also inhibited 4-hydroxymephenyloin production(CYP2C19) at the 10 and 33 μM dose levels. Inhibition of other CYP450isoforms was not observed. It should be noted that the IC50 for CYP2C9in this experiment was approximately three times that reported inExample 7 (11 μM as compared to 3.6 μM). This result is most likely due,at least in part, to the use of a lower purity (−) halofenic acid (loweree) in Example 7.

TABLE 5 Hepatic Microsomal Activities of Phenacetin (CYP1A2) andCoumarin (CYP2A6) in Male and Female Human Microsomes Incubated with (−)Halofenic Acid at Doses of 0.33, 1.0, 3.3, 10, and 33.3 μM. PhenacetinCoumarin AC 7-HC Control/ Production Production Test Conc (pmol/mg %(pmol/mg % Article (μM) protein/min) Inhibition protein/min) InhibitionCIC 33.3 0.00 ± 0.00 NA  0.00 ± 0.00 NA VC 0.1% 118 ± 2  0 32.0 ± 1.4 0FUR 5 54.5 ± 1.3  54 NA NA TRAN 250 NA NA  0.00 ± 0.00 100 (−) 0.33 116± 2  1 33.3 ± 0.7 −4 halofenic 1.0 118 ± 2  0 32.6 ± 0.7 −2 acid 3.3 119± 2  −1 32.1 ± 0.7 0 10 119 ± 2  −1 33.1 ± 0.7 −3 33.3 119 ± 2  −1 32.3± 0.7 −1 IC₅₀ NA NA Values are the mean ± standard deviation of N = 3samples (VC: N = 6). Abbreviations: Conc, concentration; AC,acetaminophen; 7-HC, 7-hydroxycoumarin; CIC, chromatographicinterference control; VC, vehicle control (0.1% methanol); NA, notapplicable; FUR, furafylline; TRAN, tranylcypromine.

TABLE 6 Hepatic Microsomal Activities of Tolbutamide (CYP2C9) and S-Mephenytoin (CYP2C19) in Male and Female Human Microsomes Incubated with(−) Halofenic Acid at Doses of 0.33, 1.0, 3.3, 10, and 33.3 μM.Tolbutamide S-Mephenytoin 4-OH TB 4-OH ME Control/ Production ProductionTest Conc (pmol/mg % (pmol/mg % Article (μM) protein/min) Inhibitionprotein/min) Inhibition CIC 33.3  0.00 ± 0.00 NA 0.00 ± 0.00 NA VC 0.1%43.0 ± 1.4 0 3.17 ± 0.29 0 OMP 10 NA NA 1.58 ± 0.05 50 SFZ 50 BQL ~100NA NA (−) 0.33 41.0 ± 0.9 5 3.03 ± 0.03 4 halofenic 1.0 38.6 ± 0.5 103.01 ± 0.07 5 acid 3.3 34.2 ± 0.2 21 2.69 ± 0.12 15 10 22.7 ± 0.6 472.43 ± 0.09 23 33.3 12.7 ± 0.2 71 1.80 ± 0.07 43 IC₅₀ ~11.335 μM >33.3μM Values are the mean ± standard deviation of N = 3 samples (VC: N =6). Abbreviations: Conc, concentration; 4-OH TB, 4-hydroxytolbutamide;4-OH ME, 4-hydroxymephenytoin; CIC, chromatographic interferencecontrol; VC, vehicle control (0.1% methanol); NA, not applicable; OMP,omeprazole; SFZ, sulfaphenazole; BQL, below quantifiable limit.

TABLE 7 Hepatic Microsomal Activities of Dextromethorphan (CYP2D6) andChlorzoxazone (CYP2E1) in Male and Female Human Microsomes Incubatedwith (−) Halofenic Acid at Doses of 0.33, 1.0, 3.3, 10, and 33.3 μM.Dextromethorphan Chlorzoxazone DEX 6-OH CZX Control/ ProductionProduction Test Conc (pmol/mg % (pmol/mg % Article (μM) protein/min)Inhibition protein/min) Inhibition CIC 33.3 0.00 ± 0.00 NA 0.00 ± 0.00NA VC 0.1% 111 ± 6  0 246 ± 5  0 4-MP 100 NA NA BQL ~100 QUIN 1 BQL ~100NA NA (−) 0.33 107 ± 4  3 238 ± 4  3 halofenic 1.0 110 ± 2  1 244 ± 1  1acid 3.3 104 ± 3  6 239 ± 4  3 10 107 ± 1  4 244 ± 6  1 33.3 106 ± 4  5239 ± 4  3 IC₅₀ NA NA Values are the mean ± standard deviation of N = 3samples (VC: N = 6). Abbreviations: Conc, concentration; DEX,dextrorphan; 6-OH CZX, 6-hydroxychlorzoxazone; CIC, chromatographicinterference control; VC, vehicle control (0.1% methanol); NA, notapplicable; 4-MP, 4-methylpyrazole; QUIN, quinidine; BQL, belowquantifiable limit.

TABLE 8 Hepatic Microsomal Activities of Testosterone (CYP3A4) in Maleand Female Human Microsomes Incubated with (−) Halofenic Acid at Dosesof 0.33, 1.0, 3.3, 10, and 33.3 μM. Control/ Testosterone Test Conc6β-OHT Production Article (μM) (pmol/mg protein/min) % Inhibition CIC33.3   0.00 ± 0.00 NA VC 0.1% 1843 ± 9 0 KTZ 5  32.4 ± 0.2 98.2 (−) 0.33 1816 ± 12 1.5 halofenic 1.0  1851 ± 14 0 acid 3.3 1810 ± 3 1.8 10 1819± 4 1.3 33.3 1816 ± 6 1.5 IC₅₀ NA Values are the mean ± standarddeviation of N = 3 samples (VC: N = 6). Abbreviations: Conc,concentration; 6β-OHT, 6β-hydroxytestosterone; CIC, chromatographicinterference control; VC, vehicle control (0.1% methanol); NA, notapplicable; KTZ, ketoconazole; BQL, below quantifiable limit.

Example 19

While one of ordinary skill in the art would understand how to assessthe ability of a compound to inhibit cyclooxygenase (COX-1), thisexample illustrates a method for assessing the ability of compounds ofthe present invention to inhibit cyclooxygenase (COX-1) as exemplifiedby (−) halofenate, an exemplary compound of the invention, andketoprofen, a positive control.

An assay for cyclooxygenase 1 (COX-1) inhibition by the compounds of thepresent invention was conducted using fresh human blood from healthydonors. For the assessment of the ability of (−) halofenate to inhibitCOX-1, (−) halofenate was added to heparinized blood prior to theactivation of COX-1. The enzyme was activated by the commerciallyavailable calcium ionophore A23187.

This bioassay is based on the production of thromboxane B2 (TXB2) afterCOX-1 activation. When activated, COX-1 generates prostaglandin H2 whichis then converted to thromboxane A2 (TXA2) by thromboxane synthase andthen finally to thromboxane B2 by non-enzymatic hydroxylation. Inaddition, PGE2 levels were measured to exclude inhibition of thromboxanesynthase. This assay allows discrimination between COX-1 and thromboxanesynthase inhibition.

TXB2 and PGE2 were measured by commercially available immunoassay kitspurchased from Assay Designs, Inc. (Ann Arbor, Mich.). All otherchemicals were from Sigma except as noted. (−) halofenate and thepositive control compound ketoprofen, were dissolved in DMSO as 100×stock solutions. Immediately before use, all compounds were diluted 1:10in RPMI-1640 medium (Gibco BRL, 11875-093) to make a 10× concentratedstock solution. The 10×RPMI-1640 stock solutions of the calciumionophore were prepared in an identical manner. In a 96-well plate, 25microliters of 10× (−) halofenate stock solution in RPMI-1640 was mixedwith 200 microliters heparinized fresh human blood for 15 minutes atroom temperature. COX-1 was then activated by adding 25 microliters ofthe 10× stock solution of 25 micromolar calcium ionophore. The plateswere shaken for 10 minutes at room temperature and then incubated at 37°C. for an additional 30 minutes. After these incubation times, theplates were centrifuged at 2,000 g for 5 minutes. Two microliters ofplasma were taken from each reaction for the measurement of TXB2 usingan enzyme immunoassay kit from Assay Designs, Inc. The PGE2 level in thesame reaction was measured using a PGE2 enzyme immunoassay kit fromAssay Designs, Inc.

The results of the assay are presented as the percent inhibition ofCOX-1 activity. The percent inhibition of COX-1 activity is definedaccording to the following equation:%Inhibition=100−[(TXB2_(treated)−TXB2_(unstimulated))/(TXB2_(untreated)−TXB2_(unstimulated))]×100%.wherein TXB2_(treated) is the TXB2 value of plasma from testcompound-treated (e.g., (−) halofenate treated), ionophore stimulatedblood, TXB2_(unstimulated) represents the level of TXB2 of plasma fromionophore unstimulated and test compound untreated blood, andTXB2_(untreated) is the TXB2 level of plasma from ionophore stimulatedblood that was not treated with an agent to inhibit the COX-1 enzyme.

Ketoprofen served as the positive control for COX-1 assay. Ketoprofeninhibited the COX-1 enzyme in blood with an IC50 of 0.03 mM. Racemichalofenate was found to inhibit the COX-1 enzyme in blood with an IC50of about 1 mM. (−) halofenate was found to inhibit the COX-1 enzyme inblood with an IC50 of greater than 3 mM (see FIG. 18). Levels of PGE2were not changed by incubation with these compounds, indicating thatthromboxane synthetase was not inhibited and the observed changes weredue to inhibition of COX-1. Thus, (−) halofenate is a substantiallyweaker and ineffective inhibitor of the COX-1 enzyme. Therapeuticadministration of the (−) halofenate should be associated with a muchlower incidence of gastrointestinal toxicity than the administration ofracemic halofenate or the corresponding (+) halofenate isomer.

The COX-1 inhibitory effects of halofenate occur at levels capable ofexplaining the adverse GI effects of halofenate. The concentration ofracemic halofenate used in clinical trials has exceeded the IC₅₀ forinhibiting the enzyme.

Although the foregoing invention has been described in detail forpurposes of clarity of understanding, it will be obvious that certainmodifications can be practiced within the scope of the appended claims.All publications and patent documents cited herein are herebyincorporated by reference in their entirety for all purposes to the sameextent as if each were so individually denoted.

1. A method of treating hyperuricemia in a mammal, comprisingadministering to said mammal a therapeutically effective amount of the(−) stereoisomer of a compound of Formula I,

wherein: R is a hydroxy group, a benzyloxy, phenethyloxy,formamidoethoxy, acetamidoethoxy, acetamidopropoxy, benzamidoethoxy,benzamidopropoxy, carbamoylmethoxy, carbamoylethoxy,2-(4-chlorophenoxy)ethoxy, 2-(4-chlorophenoxy)-2-methylpropoxy or a2-carbamoylphenoxy group forming an ester linkage capable of beinghydrolyzed upon administration to the mammal to provide a compound offormula I wherein R is OH; and each X is independently a halogen; or apharmaceutically acceptable salt thereof, wherein the compound containsthe (−) stereoisomer in an enantiomeric excess of at least 80%.
 2. Themethod of claim 1, wherein the (−) stereoisomer of the compound isselected from the group consisting of (−) 2-acetamidoethyl4-chlorophenyl-(3-trifluoromethylphenoxy)acetate and (−)4-chlorophenyl-(3-trifluoromethylphenoxy)acetic acid and thepharmaceutically acceptable salts thereof.
 3. The method of claim 2,wherein the (−) stereoisomer of the compound is administered togetherwith a pharmaceutically acceptable carrier.
 4. The method of claim 2,wherein the (−) stereoisomer is in an enantiomeric excess of at least98%.
 5. The method of claim 2, wherein the compound is administered byan intravenous, transdermal, or oral route.
 6. The method of claim 2,wherein the amount administered is about 100 mg to about 3000 mg perday.
 7. The method of claim 2, wherein the amount administered is about500 mg to about 1500 mg per day.
 8. The method of claim 2, wherein theamount administered is about 5 to about 250 mg per kg per day.
 9. Themethod of claim 2, wherein the enantiomeric excess is at least 96%. 10.The method of claim 2, wherein the therapeutically effective amount isfrom 100 to 500 mg.
 11. The method of claim 2, wherein thetherapeutically effective amount is from 500 to 1000 mg.
 12. The methodof claim 1, wherein R is selected from the group consisting ofbenzyloxy, phenethyloxy, dimethyaminoethoxy, diethylaminopropoxy,benzamidoethoxy, benzamidopropoxy, carbamoylmethoxy, carbamoylethoxy,2-(4-chlorophenoxy)ethoxy, 2-(4-chlorophenoxy)-2-methylpropoxy and2-carbamoylphenoxy.
 13. The method of claim 1, wherein hydrolysis of theester provides (−) 4-chlorophenyl-(3-trifluoromethylphenoxy)acetic acid.